CN103820346A - Brew yeast and application of brew yeast to manufacturing alcohol through fermentation - Google Patents
Brew yeast and application of brew yeast to manufacturing alcohol through fermentation Download PDFInfo
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- CN103820346A CN103820346A CN201410088818.3A CN201410088818A CN103820346A CN 103820346 A CN103820346 A CN 103820346A CN 201410088818 A CN201410088818 A CN 201410088818A CN 103820346 A CN103820346 A CN 103820346A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 30
- 230000004151 fermentation Effects 0.000 title claims abstract description 30
- 238000004519 manufacturing process Methods 0.000 title abstract description 14
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 38
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- 239000008103 glucose Substances 0.000 claims description 32
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- 229940041514 candida albicans extract Drugs 0.000 claims description 20
- 239000012138 yeast extract Substances 0.000 claims description 20
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- 229910001628 calcium chloride Inorganic materials 0.000 claims description 10
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- 229910017053 inorganic salt Inorganic materials 0.000 claims description 8
- 239000002054 inoculum Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 238000012262 fermentative production Methods 0.000 claims description 6
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical group [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 5
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- 239000001888 Peptone Substances 0.000 claims description 4
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- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 description 2
- 229960000907 methylthioninium chloride Drugs 0.000 description 2
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- 229910052757 nitrogen Inorganic materials 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
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- 229960003487 xylose Drugs 0.000 description 1
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- 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
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a strain of brew yeast and application of the brew yeast to manufacturing alcohol through fermentation. The brew yeast, (Saccharomyces cerevisiae) SC-X234, is collected on January 6th, 2014 at China Center for Type Culture Collection (CCTCC), with the collection number of CCTCC M2014005; the feeble train N<+> infusion mediate spt15 gene technology is adopted, SC-100 is used as initiation strain; primary screening is conducted by utilizing a TTC slab and an alcohol slab; the obtained strain is subjected to fermentation and secondary screening to gradually increase the tolerance of the yeast in alcohol; the manufacturing strain SC-X234 with the yield of 15.75% can be finally obtained; the strain can greatly increase the fermentation productivity; the sugar to alcohol conversion ratio is high; the alcohol yield in a 5 L fermentation tank can reach 126 g/L, which is 87.5% higher than that of the initiation strain; the alcohol has a very high economic value as a fuel alcohol.
Description
Technical field
The invention belongs to microbial fermentation technology field, be specifically related to an Accharomyces cerevisiae and the application in fermentation producing and ethanol thereof.
Background technology
Fossil energy is the main body of current energy structure, is the Nonrenewable resources that a class is very valuable, and its reserves are limited, take China as example, though China is oil production big country, is oil consumption big country and petroleum resources shortage big country also.Within 2008, China produces 1.9 hundred million tons of crude oil; 3.65 hundred million tons, consumption oil, 1.75 hundred million tons of net importation crude oil, import volume accounts for 48% of total quantity consumed.And by the end of the year 2006, it is 20.43 hundred million tons that oil workable reserve is verified in China's accumulation, calculate like this, existing oil only can maintain the needs of decades, and contradiction between oil supply and demand is very outstanding, and will constantly aggravate.Under this overall situation, carry out the exploitation of biomass energy, replace non-renewable fossil energy just to there is important practical significance.The Chinese government has also been fully recognized that the importance of application this " green energy resource " at present, in recent years the investment of biomass energy research is constantly increased, and particularly research and development is substituted to grain-production alcohol fuel with vegetable fibre and gives great expectations.
Alcohol fuel (fuel ethanol) is to point in gasoline or diesel oil to add a certain proportion of dehydrated alcohol.One solves the potential a limited number of problems as primary energy source gasoline, diesel oil; The 2nd, the burning level of raising gasoline and diesel oil, is conducive to strengthen environmental protection.Although the fuel value of alcohol is only 2/3 of gasoline combustion value, in alcohol molecule, containing aerobic, in combustion processes, the capability of antidetonance is good.In the time that the addition of alcohol in gasoline is no more than 15%, the rideability of automobile is had no significant effect, but the content of CO, NOx compound and CH compound in tail gas can reduce by 30% ~ 50%.On technical research, countries in the world have all been dropped into huge manpower and materials and have been carried out the exploitation of alcohol fuel, especially research and develop by the alternative grain-production alcohol fuel advantage of vegetable fibre remarkable.Take the U.S. as example, the U.S. is with the morning of technological development of Mierocrystalline cellulose ethanol processed, USDOE is submitted a plan for 1999, by 2005, the production cost of alcohol fuel is reduced to 36%, 1999, renewable energy source institute of the U.S. and about exploitation enterprise crucial fermentation technique reached quite high level, certainly remain in some problems.Various countries scientist has obtained major progress in a lot of fields at present: as the production efficiency of microbial cellulase significantly improves, multiple new raw materials pretreatment practical technique is developed; The engineering bacteria of glucose fermentation and wood sugar successfully builds altogether; The research of straw biological transforming fuel alcohol reactor obtains certain progress; The pilot scale of cellulose raw producing and ethanol has been extended to tens of cubic metres of fermentor tank industrial scales etc.Utilize agricultural crop straw and the forest products tankage (lignocellulose-containing) of enormous amount to produce alcohol fuel, listed " 15 ", " 11th Five-Year " development in science and technology of China in the works.Utilizing cellulosic material to produce ethanol is to utilize the microorganism of cellulase-producing or cellulase first cellulose hydrolysis to be become to fermentable sugar, recycling yeast is fermented into ethanol, therefore the bacterial strain that obtains efficient producing and ethanol is key factor wherein, and various screening high-yield ethanol bacterial strains become the focus of research simultaneously.Improve by having introduced a kind of novel mutafacient system the ability that bacterial strain produces alcohol herein, improved the patience of yeast saccharomyces cerevisiae to ethanol simultaneously.The highest level of current domestic producing and ethanol is 12%, and external highest level can arrive 15%.
Summary of the invention
Low in order to solve in current fermentation producing and ethanol ethanol production, there is no the bacterial strain of high-yield ethanol, and then the high problem of fermentation producing and ethanol production cost, the invention provides an Accharomyces cerevisiae SC-X234 and the application in fermentation producing and ethanol thereof, improve the patience of yeast saccharomyces cerevisiae to alcohol in fermentation by mutagenesis, guaranteed that ethanol production is in 15.75% level simultaneously.
In order to achieve the above object, the technical solution used in the present invention is: a kind of yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) SC-X234, be preserved in Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC M 2014005, address: Wuhan, China Wuhan University on January 6th, 2014.
The screening method of Saccharomyces Cerevisiae in S C-X234 of the present invention: the original starting strain SC-100 of yeast saccharomyces cerevisiae is after ion beam of low energy N+ injects mediation spt15 gene, the bacterial strain that utilizes high ethanol flat board, TTC plate screening to obtain, after the domestication of resistance to ethanol and shake flask fermentation to obtain the yeast saccharomyces cerevisiae that producing and ethanol concentration is the highest be next round mutagenic strain.Repeat above-mentioned steps, until obtain aimed strain SC-X234, concrete steps are as follows:
(a) preparation of monospore suspension: the yeast saccharomyces cerevisiae bacterium SC-100 that sets out is made to spore suspension, and concentration is 1 × 10
6individual/mL;
(b) mutagenesis of Mediated by Low Energy Ion Beam spt15 gene: get 100 μ L spore suspensions and the 50 μ L plasmid (being permitted beautiful jade teacher containing the plasmid of spt15 by Nanjing University of Technology gives) containing spt15, evenly be applied on aseptic empty flat board, dry up with sterile wind, under 20Kev energy, 40 × 10
14ions/cm
2under dosage, carry out the mutagenesis of nitrogen ion, ion implantation after, under gnotobasis, carry out wash-out with 1mL TE solution, the bacterium liquid after wash-out 37 ℃ temperature bathe 1h, be then applied on alcohol flat board, carry out pressure screening, 37 ℃ be inverted cultivate 2-3d.
(c) primary dcreening operation of TTC flat board: TTC upper strata substratum low temperature is poured on the flat board of having grown in step (b), is inverted and cultivates 1-2d at 37 ℃, screen the bacterial strain that cell dehydrogenase activity is strong, vigor is stronger, and redness is darker.
(d) the multiple sieve of fermentation: the S. cervisiae access inclined-plane that step (c) is filtered out, at 37 ℃, cultivate 1-2d, then inclined-plane proceeds in seed culture medium, at 37 ℃, cultivates 8-10h.Get in seed liquor access fermention medium, inoculum size 10%(v/v), 250mL shaking flask dress 30mL substratum, 37 ℃ of leavening temperatures, fermentation 48h, measure the content of alcohol in fermented liquid, filter out S. cervisiae that ethanol content the is the highest starting strain as next round mutagenesis screening, repeat above-mentioned steps until screen aimed strain SC-X234.
In above-mentioned screening method: the alcohol flat board in step (b) is the alcohol that adds 5%-10% in YPD substratum, the TTC substratum of step (c) is divided into upper strata substratum and lower floor's substratum, lower floor's substratum is YPD substratum, upper strata nutrient media components be TTC 0.5g glucose 5g agar 15g constant volume to 1L, pH nature.Step (d) seed culture based component carbon source 5%, nitrogenous source 0.7%, inorganic salt 0.016%, buffer reagent 0.1 %, pH=5.5, fermentation culture based component composition in step (d): carbon source 36%, nitrogenous source 1.1%, inorganic salt 0.02%, buffer reagent 0.5%, wherein carbon source is one or the mixture in glucose or W-Gum; Nitrogenous source: organic nitrogen source is yeast extract paste, inorganic nitrogen-sourced: ammonium sulfate, pH=5.5, buffer reagent is primary ammonium phosphate.
Yeast saccharomyces cerevisiae of the present invention (
saccharomyces cerevisiae) morphology and the plysiochemical characteristic of SC-X234:
Colony colour: oyster white
Aerobic mode: amphimicrobian
Bacterium colony size: 2-3mm
Growth temperature: 35-40 ℃
Optimal pH: 5.0-5.5
Colonial morphology: circle
Methylene blue dyeing: viable bacteria is colourless, dead bacterium is blue.
The application of above-mentioned Saccharomyces Cerevisiae in S C-X234 in fermentative production of ethanol, comprises the steps:
1) dull and stereotyped cultivation: Saccharomyces Cerevisiae in S C-X234 is connected on dull and stereotyped minimum medium and is cultivated, and culture temperature is 35-40 ℃, incubation time 1-2d;
2) slant culture: the Saccharomyces Cerevisiae in S C-X234 that step 1) middle plateform is cultivated is transferred on inclined-plane minimum medium, incubation time 18-20h, culture temperature 35-40 ℃;
3) planting liquid cultivates: by step 2) in the Saccharomyces Cerevisiae in S C-X234 of slant culture receive in seed culture medium and cultivate, incubation time 8-10h, culture temperature 35-40 ℃, shaking speed is 150r/min;
4) fermentation culture: the kind liquid in step 3) is transferred in fermention medium, and inoculum size is 10%, and incubation time is 48h, and culture temperature is 35-40 ℃, and shaking speed is 100r/min.
The component that minimum medium wherein: step 1) and 2) comprises following mass percent: carbon source 2%, nitrogenous source 3-4%, agar 2%, all the other are water, pH nature; Wherein said carbon source is one or both in glucose or W-Gum, and described nitrogenous source is the mixture of peptone or yeast powder.
The component that step 3) seed culture medium comprises following mass percent: carbon source 5%, nitrogenous source 0.5-1%, inorganic salt 0.01-0.02%, buffer reagent 0.1-0.2%, pH=5.5, wherein carbon source is one or more mixtures in glucose, W-Gum or molasses; Nitrogenous source is one or more in yeast powder, ammonium sulfate or Secondary ammonium phosphate, and buffer reagent is primary ammonium phosphate.
The component that step 4) fermention medium comprises following mass percent: carbon source 32-36%, nitrogenous source 1-2%, inorganic salt 0.02-0.05%, buffer reagent 0.5-1%, pH=5.5, wherein carbon source is one or more mixtures in glucose or W-Gum; Nitrogenous source is one or more in yeast extract paste, ammonium sulfate, and buffer reagent is primary ammonium phosphate.
Injecting mediation spt15 gene is in order to improve the patience of yeast saccharomyces cerevisiae to ethanol, spt15 is hansen initiation transcription factor, the sudden change of spt15 gene can make the alcohol tolerance of yeast saccharomyces cerevisiae change, and in order to screen the yeast saccharomyces cerevisiae of high resistance to ethanol, thereby obtains the yeast saccharomyces cerevisiae of high-yield ethanol.
Beneficial effect: the present invention has adopted low energy N
+inject mediation spt15 gene engineering, with SC-100 for the bacterium that sets out, utilize TTC flat board, alcohol flat board to carry out primary dcreening operation, the bacterial strain the obtaining multiple sieve that ferments, improve gradually the patience of yeast in alcohol, finally obtain output and can reach 15.75% production bacterial strain SC-X234, this bacterial strain can improve the problem that fermentation production rate is low greatly, and sugar alcohol transformation efficiency is higher.In 5L fermentor tank, ethanol production can reach 126g/L, has improved 87.5% than the original bacterium that sets out, and has very high economic worth as alcohol fuel.
Embodiment
According to following embodiment, the present invention may be better understood.But, those skilled in the art will readily understand, the described concrete material proportion of embodiment, processing condition and result thereof be only for the present invention is described, and should also can not limit the present invention described in detail in claims.
The screening method of embodiment 1 Saccharomyces Cerevisiae in S C-X234
Starting strain Saccharomyces Cerevisiae in S C-100 is convenient to mark purchased from the Angel Yeast dry powder in Angel Yeast stock company, and laboratory is by its called after SC-100.
With SC-100, for the bacterium screening Saccharomyces Cerevisiae in S C-X234 that sets out, concrete steps are as follows:
A) preparation of monospore suspension: the yeast saccharomyces cerevisiae bacterium that sets out is made to spore suspension, and concentration is 1 × 10
6individual/mL.
B) mutagenesis of Mediated by Low Energy Ion Beam spt15 gene: get 100 μ L spore suspensions and the 50 μ L plasmid containing spt15, be evenly applied on aseptic flat board, dry up with sterile wind, under 20Kev energy, 40 × 10
14ions/cm
2under dosage, carry out the mutagenesis of nitrogen ion, ion implantation after, under gnotobasis, carry out wash-out with 1mL TE solution, the bacterium liquid after wash-out 37 ℃ temperature bathe 1h, be then applied on alcohol flat board, carry out pressure screening, 37 ℃ be inverted cultivate 2-3d.
C) screening of mutagenic fungi
The primary dcreening operation of TTC flat board: TTC upper strata substratum low temperature is poured on the flat board of having grown in step (b), is inverted and cultivates 1-2d at 37 ℃, screen the bacterial strain that cell viability is strong, dehydrogenase activity is stronger, and redness is darker.
D) the multiple sieve of fermentation: in the S. cervisiae access slant medium that step (c) is filtered out, cultivate 1-2d at 37 ℃, then inclined-plane proceeds in kind of liquid, cultivates 8-10h at 37 ℃.Get in seed liquor access fermention medium, inoculum size 10%(v/v), 250mL shaking flask dress 30mL substratum, rotating speed is 100r/min, 37 ℃ of leavening temperatures, fermentation 48h, the content of alcohol in mensuration fermented liquid, filter out S. cervisiae that ethanol content the is the highest starting strain as next round mutagenesis screening, repeat above-mentioned steps until screen aimed strain SC-X234.
Wherein culture medium prescription is:
The alcohol plate culture medium of step in b) is: all the other are water pH nature for glucose 2%, fish meal protein peptone 2%, agar 2%, yeast extract paste 2%, alcohol 8%.The TTC substratum of step in c) is: TTC0.5g, glucose 5g, agar 15g constant volume are to 1L nature pH
The TTC flat board of step (c) is divided into upper strata substratum and lower floor's substratum, and lower floor's substratum is YPD substratum, upper strata nutrient media components be TTC 0.5g glucose 5g agar 15g constant volume to 1L, pH nature.
Seed liquor substratum in step d): glucose 5%, yeast extract paste 0.5%, magnesium sulfate 0.01%, calcium chloride 0.06g/L, ammonium sulfate 0.2%, Secondary ammonium phosphate 0.1%, pH are 5.5, wherein glucose need to divide disappear (divide and disappear for separating the meaning of independent sterilizing).
Fermention medium in step d): glucose 36%, yeast extract paste 0.1%, calcium chloride 0.01%, ammonium sulfate 1%, Secondary ammonium phosphate 0.5%, magnesium sulfate 0.01%, pH5.5, wherein glucose need to divide and disappears.
Fermentation results ethanol content detects as table 1
Table 1
| Bacterium number | Bacterium SC-100 sets out | Mutagenic fungi SC-X234 |
| Ethanol content (v/v) | 8% | 15% |
Have greatly improved through the ability of the product alcohol of the yeast of the mutagenic and breeding bacterium that sets out.
Embodiment 2
Biology morphology and the genetic stability of the present embodiment explanation mutagenic fungi Saccharomyces Cerevisiae in S C-X234
The morphology of Saccharomyces Cerevisiae in S C-X234 of the present invention and plysiochemical characteristic: bacterium colony is creamy white, amphimicrobian, bacterium colony size is 2-3mm, circular bacterium colony, growth temperature 35-40 ℃, optimal pH is 5.0-5.5, methylene blue dyeing: viable bacteria is colourless, dead bacterium is blue.
With the experiment of going down to posterity of the substratum in embodiment 1 and culture condition, result is as table 2,
The genetic stability of table 2 Saccharomyces Cerevisiae in S C-X234
| Passage number | Alcohol output (v/v) |
| 1 | 15.25 |
| 2 | 15 |
| 3 | 15 |
| 4 | 15.25 |
| 5 | 14.75 |
| 6 | 15.25 |
From genetic stability interpretation, mutant strain Saccharomyces Cerevisiae in S C-X234 is through 6 experiments of going down to posterity, and its alcohol output is stable, has good genetic stability, can be used as the bacterial strain of further R and D.
Embodiment 3
This example explanation Saccharomyces Cerevisiae in S C-X234 utilizes corn powder saccharification liquid fermentation to produce alcohol
Described in the present embodiment, culture medium prescription is as follows:
Plate culture medium: glucose 2%, peptone 2%, agar 2%, yeast extract paste 2%, all the other are water, natural pH.
Slant medium: all the other are water for glucose 2%, peptone 2%, agar 2%, yeast extract paste 2%, natural pH.
Seed culture medium: glucose 5%, yeast extract paste 0.5%, magnesium sulfate 0.01%, calcium chloride %, ammonium sulfate 0.2%, Secondary ammonium phosphate 0.1%, pH are 5.5, wherein glucose need to divide and disappears.
Fermention medium: corn powder saccharification liquid 32%, yeast extract paste 0.1%, calcium chloride 0.01%, ammonium sulfate 1%, Secondary ammonium phosphate 0.5%, magnesium sulfate 0.01% pH5.5, wherein glucose need to divide and disappears, and corn powder saccharification liquid is that corn is through α-amylase and saccharifying enzyme product after treatment.
Saccharomyces Cerevisiae in S C-X234 is inoculated into plate culture medium, 37 ℃ of culture temperature, incubation time 12h-24h, then proceeds to slant medium, at 37 ℃, cultivates 1-2d, and then inclined-plane proceeds in seed culture medium, at 37 ℃, cultivates 8-10h.Get in seed liquor access fermention medium inoculum size 10%(v/v), 250mL shaking flask dress 30mL substratum, rotating speed is 100r/min, 37 ℃ of leavening temperatures, fermentation 48h, the content of measuring alcohol in fermented liquid reaches 13.5%(v/v), improve 50.6% than the original bacterium that sets out of equal conditions.
Embodiment 4
This example explanation mutant strain SC-X234 yeast saccharomyces cerevisiae utilizes molasses fermented product alcohol
This tests described experiment culture medium prescription
Plate culture medium: glucose 2%, fish meal protein peptone 2%, agar 2%, yeast extract paste 2%, all the other are water, natural pH.
Slant medium: glucose 2%, fish meal protein peptone 2%, agar 2%, yeast extract paste 2%, all the other are water, natural pH.
Seed culture medium: glucose 5%, yeast extract paste 0.5%, magnesium sulfate 0.01%, calcium chloride 0.06%, ammonium sulfate 0.2%, Secondary ammonium phosphate 0.1%, pH are 5.5, wherein glucose need to divide and disappears.
Fermention medium: molasses 32%, yeast extract paste 0.1%, calcium chloride 0.01%, ammonium sulfate 1%, Secondary ammonium phosphate 0.5%, magnesium sulfate 0.01%, pH5.5, wherein glucose need to divide and disappears.
Saccharomyces Cerevisiae in S C-X234 is inoculated into plate culture medium, 37 ℃ of culture temperature, incubation time 12h-24h, then proceeds to slant medium, at 37 ℃, cultivates 1-2d, and then inclined-plane proceeds in kind of liquid, at 37 ℃, cultivates 8-10h.Get in seed liquor access fermention medium inoculum size 10%(v/v), 250mL shaking flask dress 30mL substratum, rotating speed is 100r/min, 37 ℃ of leavening temperatures, fermentation 48h, the content of measuring alcohol in fermented liquid reaches 10.5%(v/v), improve 75% than the original bacterium that sets out of equal conditions.
Embodiment 5
This example explanation mutant strain SC-X234 yeast saccharomyces cerevisiae produces alcohol in 5L fermentation cylinder for fermentation
This tests described experiment culture medium prescription
Plate culture medium: all the other are water for glucose 2%, fish meal protein peptone 2%, agar 2%, yeast extract paste 2%, natural pH.
Slant medium: all the other are water for glucose 2%, fish meal protein peptone 2%, agar 2%, yeast extract paste 2%, natural pH.
Seed culture medium: glucose 5%, yeast extract paste 0.5%, magnesium sulfate 0.01%, calcium chloride 0.06%, ammonium sulfate 0.2%, Secondary ammonium phosphate 0.1%, pH are 5.5, wherein glucose need to divide and disappears.
Fermention medium: glucose 32%, yeast extract paste 0.1%, calcium chloride 0.01%, ammonium sulfate 1%, Secondary ammonium phosphate 0.5%, magnesium sulfate 0.01%, pH5.5, wherein glucose need to divide and disappears.
Saccharomyces Cerevisiae in S C-X234 is inoculated into plate culture medium, 37 ℃ of culture temperature, incubation time 12h-24h, then proceeds to slant medium, at 37 ℃, cultivates 1-2d, and then inclined-plane proceeds in kind of liquid, at 37 ℃, cultivates 8-10h.Get seed liquor and access in the 5L fermentor tank that contains 3L fermention medium, inoculum size 10%(v/v), rotating speed is 100r/min, 37 ℃ of leavening temperatures, fermentation 48h, detects ethanol content and reaches 15.75%, and the starting strain under the same terms has improved 96.9% on year-on-year basis.
In above embodiment, alcohol detection method is: chromatography of gases (capillary column AC-20) post case temperature: 60 ℃; Sampler temperature: 190 ℃; Detector temperature: 240 ℃.Internal standard substance: n-propyl alcohol.Detect liquid collocation method: (0.5mL 6% n-propyl alcohol solution+0.5mL sample) is settled to 10mL.Sample size: 2 μ L.
Per-cent in culture medium prescription of the present invention (%) is mass percent.
Claims (6)
- A yeast saccharomyces cerevisiae ( saccharomyces cerevisiae) SC-X234, be preserved in Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC M 2014005.
- 2. the application of Saccharomyces Cerevisiae in S C-X234 claimed in claim 1 in fermentative production of ethanol.
- 3. the application of Saccharomyces Cerevisiae in S C-X234 according to claim 2 in fermentative production of ethanol, is characterized in that comprising the steps:1) dull and stereotyped cultivation: Saccharomyces Cerevisiae in S C-X234 is connected on dull and stereotyped minimum medium and is cultivated, and culture temperature is 35-40 ℃, incubation time 1-2d;2) slant culture: the Saccharomyces Cerevisiae in S C-X234 that step 1) middle plateform is cultivated is transferred on inclined-plane minimum medium, incubation time 18-20h, culture temperature 35-40 ℃;3) planting liquid cultivates: by step 2) in the Saccharomyces Cerevisiae in S C-X234 of slant culture receive in seed culture medium and cultivate, incubation time 8-10h, culture temperature 35-40 ℃;4) fermentation culture: the kind liquid in step 3) is transferred in fermention medium, and inoculum size is 10%, and incubation time is 48h, and culture temperature is 35-40 ℃.
- 4. the application of Saccharomyces Cerevisiae in S C-X234 according to claim 3 in fermentative production of ethanol, it is characterized in that: step 1) and 2) in the minimum medium component that comprises following mass percent: carbon source 2%, nitrogenous source 3-4%, agar 2%, all the other are water, pH nature; Wherein said carbon source is one or both in glucose or W-Gum, and described nitrogenous source is peptone or yeast extract paste.
- 5. the application of Saccharomyces Cerevisiae in S C-X234 according to claim 3 in fermentative production of ethanol, it is characterized in that: the component that step 3) seed culture medium comprises following mass percent: carbon source 5%, nitrogenous source 0.5-1%, inorganic salt 0.01-0.02%, buffer reagent 0.1-0.2%, pH=5.5, wherein carbon source is one or more mixtures in glucose, W-Gum or molasses; Nitrogenous source is one or more in yeast extract paste, ammonium sulfate or Secondary ammonium phosphate; Inorganic salt are one or more in magnesium sulfate and calcium chloride; Buffer reagent is primary ammonium phosphate.
- 6. the application of Saccharomyces Cerevisiae in S C-X234 according to claim 3 in fermentative production of ethanol, it is characterized in that: the component that step 4) fermention medium comprises following mass percent: carbon source 32-36%, nitrogenous source 1-2%, inorganic salt 0.02-0.05%, buffer reagent 0.5-1%, pH=5.5, wherein carbon source is one or more mixtures in glucose, molasses or W-Gum; Nitrogenous source is one or more in yeast extract paste, ammonium sulfate, and buffer reagent is primary ammonium phosphate, and inorganic salt are one or more in magnesium sulfate and calcium chloride.
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| 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 |
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