CN101880636B - Bacterial strain tolerant with various inhibitors of Saccharomyces cerevisiae - Google Patents
Bacterial strain tolerant with various inhibitors of Saccharomyces cerevisiae Download PDFInfo
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- CN101880636B CN101880636B CN201010192303XA CN201010192303A CN101880636B CN 101880636 B CN101880636 B CN 101880636B CN 201010192303X A CN201010192303X A CN 201010192303XA CN 201010192303 A CN201010192303 A CN 201010192303A CN 101880636 B CN101880636 B CN 101880636B
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Abstract
The invention discloses a bacterial strain tolerant with various inhibitors of Saccharomyces cerevisiae, which is named as Saccharomyces cerevisiae YYJ003 and is collected in China Committee for Culture Collection of Microorganisms (CCCCM) with the collection number of CGMCC NO.2757. The bacterial strain tolerant with the various inhibitors of the Saccharomyces cerevisiae in the invention can be in normal growth in a culture medium containing inhibitors produced by diluted acid pretreatment and can realize efficient ethanol production. The bacterial strain in the invention breaks through the bottleneck problem that a dilute acid pretreatment method is limited to produce cellulosic ethanol.
Description
Technical field
The present invention relates to a kind of yeast saccharomyces cerevisiae, particularly relate to the bacterial strain that a kind of yeast saccharomyces cerevisiae tolerates multiple suppressor factor.
Background technology
In cellulosic ethanol production technology, the acid system pre-treatment receives the favor of most mechanisms because it is with low cost and workable, is considered to easy industrialization realization pretreatment process, like dilute acid pretreatment and steam explosion pre-treatment.Yet, because the condition of these Chemical Pretreatment generally all is a HTHP, thus the generation of some inhibitory substances can be caused, like furans, weak acid class, phenols etc.These inhibitory substances have very had strong inhibitory effects to follow-up organism of fermentation, have a strong impact on the ethanol fermentation productive rate and the speed of cellulosic hydrolysate.Existing these suppressor factor of experiment proof suppress multiple organism of fermentation, comprise intestinal bacteria, zymomonas mobilis, pichia stipitis and yeast saccharomyces cerevisiae etc., and wherein, yeast saccharomyces cerevisiae is one of bacterial classification that wherein tolerance is the strongest.In addition, yeast saccharomyces cerevisiae is a most widely used bacterial strain in ethanol fermentation, and the advantage of yeast saccharomyces cerevisiae comprises: the conversion rate of (1) glucose is fast, and (2) product specificity is high, and (3) have robustness etc. to industrial production environment.
The screening and the structure that can tolerate the bacterial strain of the suppressor factor in the cellulosic hydrolysate are major issues in the cellulosic ethanol production research.There are some researches show that in early days these suppressor factor mainly suppress microbial growth, and to little many of the influence of the influence of fermentation capacity comparison growth.People hope to obtain to tolerate the Wine brewing yeast strain of multiple suppressor factor and realize original position detoxification and efficient fermentation in the cellulosic ethanol production, because the detoxification step can obviously increase production costs of cellulosic ethanol, reduce the market competitiveness of cellulosic ethanol.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of bacterial strain of the multiple suppressor factor of tolerance of yeast saccharomyces cerevisiae is provided.
Technical scheme of the present invention is summarized as follows:
A kind of bacterial strain of the multiple suppressor factor of tolerance of yeast saccharomyces cerevisiae; Its called after yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) YYJ003; Be called for short YYJ003; Delivered and be preserved in the China Committee for Culture Collection of Microorganisms common micro-organisms center of Beijing on November 21st, 2008, preserving number is CGMCC NO.2757.
Normal growth in the substratum of the suppressor factor that this bacterial strain produces in containing multiple cellulosic ethanol production.
Said suppressor factor is furfural, phenol and acetate.
The content of said suppressor factor furfural is 1.3g/L, and the content of phenol is 0.5g/L, and the content of acetate is 5.3g/L.
In cellulosic ethanol production technology, the dilute acid pretreatment method is considered to the most potential Mierocrystalline cellulose pretreatment process, but owing to the generation of suppressor factor and to the toxic action of mikrobe the widespread use of this method is restricted.The bacterial strain of the multiple suppressor factor of tolerance of a kind of yeast saccharomyces cerevisiae of the present invention can be in the substratum that contains the suppressor factor that dilute acid pretreatment produces normal growth and realize alcohol production efficiently.Bacterial strain of the present invention has been broken through a bottleneck problem of restriction dilute acid pretreatment method production of cellulosic ethanol.
Description of drawings
Fig. 1 is the colonial morphology of YYJ003 bacterial strain;
Fig. 2 is the cellular form of YYJ003 bacterial strain;
Fig. 3 is shaking YYJ003 bacterial strain and the growth curve of original S bacterial strain under mixed inhibitor on the bottle;
Fig. 4 is YYJ003 bacterial strain and the fermentation growth curve of original S bacterial strain in the low sugar culture-medium of include mixed suppressor factor;
Fig. 5 is YYJ003 bacterial strain and sugar consumption and the ethanol curve of original S bacterial strain in the low sugar culture-medium of include mixed suppressor factor;
Fig. 6 is YYJ003 bacterial strain and the furfural conversion curve of original S bacterial strain in the low sugar culture-medium of include mixed suppressor factor;
Fig. 7 is YYJ003 bacterial strain and the fermentation growth curve of original S bacterial strain in the high glucose medium of include mixed suppressor factor;
Fig. 8 is YYJ003 bacterial strain and sugar consumption and the ethanol curve of original S bacterial strain in the high glucose medium of include mixed suppressor factor;
Fig. 9 is YYJ003 bacterial strain and the furfural conversion curve of original S bacterial strain in the high glucose medium of include mixed suppressor factor.
Embodiment
In order fully to disclose the bacterial strain of the multiple suppressor factor of tolerance of a kind of yeast saccharomyces cerevisiae of the present invention, explain in conjunction with embodiment, but do not represent limitation of the present invention.
Embodiment 1
The separation of YYJ003 bacterial strain
The new bacterial strain YYJ003 bacterial strain of the multiple suppressor factor of tolerance of an Accharomyces cerevisiae of the present invention obtains through multiple suppressor factor tolerance domestication after industrial saccharomyces cerevisiae (Saccharomyces cerevisiae) is carried out ultraviolet mutagenesis.The ultraviolet mutagenesis strategy is: with nutrient solution (the YEPD substratum: glucose 20g/L of original S bacterial strain; Yeast powder 10g/L; Peptone 20g/L) is laid on the screening culture medium (adding the YEPD substratum of suppressor factor) that contains suppressor factor after diluting; The flat board of completing apart from 30cm place irradiation under the 15W uv lamp 60 seconds, is put in 30 ℃ of incubators lucifuge and cultivated 3 days.Bacterial strain domestication step is: the bacterium colony that grows on the screening culture medium is transferred to the liquid screening that contains suppressor factor selects to cultivate in the substratum, culture condition is 30 ℃, 180rpm.After being cultured to stationary phase, press OD
600=0.1 is seeded to and proceeds in another new liquid selective medium that suppressor factor is arranged to cultivate.When bacterial strain is forwarded in the new substratum, increases inhibitor concentration gradually and reach the amount in the plain dilute acid hydrolysis liquid of true fiber to inhibitor content.From the nutrient solution after the domestication, separate obtaining bacterial strain YYJ003, its colonial morphology is as shown in Figure 1, and its cellular form is as shown in Figure 2.
Embodiment 2
Shaking the growth of comparing YYJ003 bacterial strain and original strain on the bottle
1, test materials: bacterial strain YYJ003 is by this laboratory screening and be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC NO.2757; Original strain Saccharomyces cerevisiae is called for short the S bacterial strain available from Hubei Angel Yeast ltd.
2, TP:
Seed culture medium:
YYJ003 bacterial strain: glucose 20g/L, yeast powder 10g/L, peptone 20g/L, furfural 1.3g/L, phenol 0.5g/L, acetate 5.3g/L, 121 ℃ of sterilization 20min.Original strain: grape sugar 20g/L, yeast powder 10g/L, peptone 20g/L, 121 ℃ of sterilization 20min.
Fermention medium:
Glucose 20g/L, yeast powder 10g/L, peptone 20g/L, 121 ℃ of sterilization 20min.Add suppressor factor before the fermentation and make its final concentration reach furfural 1.3g/L, phenol 0.5g/L, acetate 5.3g/L.
YYJ003 bacterial strain and original strain are inoculated in respectively in the 100mL seed culture medium, cultivate 12-14h at 30 ℃, 180rpm, all with initial cell concentration OD
600=0.1 is inoculated in the 250mL Erlenmeyer flask of 100mL fermention medium, under 30 ℃, 180rpm condition, cultivates, and uses 722 type spectrophotometric determination thalli growth curves.
3, test-results:
As shown in Figure 3, the YYJ003 bacterial strain shows quick growth beginning to get into logarithmic phase through behind the 23h, and during to 48h, it is fairly obvious to grow, and changes over to stationary phase gradually, and about 80h grows into stationary phase.And original strain does not all show obvious growth trend in whole fermentation process, and until 118 hours, thalline was not spent lag phase yet.Hence one can see that, and the YYJ003 bacterial strain is containing under the condition of suppressor factor, shows fairly obvious growth vigor, compares with original strain, and it more can adapt to the environment that contains suppressor factor.
4, conclusion:
Containing under suppressor factor (concentration as stated) condition, original strain does not show obvious growth trend, and YYJ003 can adapt to the suppressor factor environment, is implemented in the growth under the suppressor factor existence.
Embodiment 3
The leavening property that compares YYJ003 bacterial strain and original strain in the 2% dextrose culture-medium fermentor tank
1, test materials: with embodiment 2.
2, TP:
The preparation of seed, fermention medium is with embodiment 2.
YYJ003 bacterial strain and original strain are inoculated in respectively in the 100mL seed culture medium, cultivate 12-14h at 30 ℃, 180rpm, all with initial cell concentration OD
600=0.1 is inoculated in the 5L fermentation tube of 3L fermention medium, under 30 ℃, 300rpm condition, cultivates, and measures thalli growth curve, glucose concn, alcohol concn and furfural concentration.
3, analytical procedure:
Cell concentration with 722 type spectrophotometric determinations its light absorption value characterizes at the 600nm place.Glucose concn, alcohol concn are measured with performance liquid chromatography (Waters1515), and chromatographic column is Aminex HPX-87H, column temperature: 65 ℃; Detector: Waters differential detector 2421; Moving phase is the 5mM sulphuric acid soln, and flow velocity 0.6mL/min, sample size are 10 μ L.Furfural concentration is also measured with performance liquid, and chromatographic column is the C18 post, column temperature: room temperature, and detector: blue rich UV-detector, the detection wavelength is 254nm, moving phase is methyl alcohol: water=6: 4, flow velocity 0.6mL/min, sample size are 10 μ L.
4, method of calculation:
Ethanol content (g/L)=(sample peak area * concentration of standard solution)/standardized solution peak area * 100%;
Alcohol yied (g/g)=record ethanol content/(amount of consumption sugar * 0.51)
Glucose and furfural content calculate same ethanol content.
5, test-results
By shown in Figure 4, when in fermentor tank, fermenting, compare the very good fermentation character of YYJ003 performance with original strain.Through about 10h, YYJ003 has just got into logarithmic phase, and shows the speed of growth faster, to 22h, grows into stationary phase, and fermenting process finishes basically.Original strain is then different, and through about 50h, it just shows certain growth trend in fermentor tank, and the speed of growth and unhappy, does not get into logarithmic phase fully.During to 120h, thalli growth speeds up, and shows tangible logarithmic growth state.Until about 135h, it grows into stationary phase, and fermenting process finishes gradually.
Can know that by Fig. 5 YYJ003 obviously is better than original strain S to the ability of utilizing of glucose.Through about 20h, YYJ003 can be with glucose completely consumed in the nutrient solution.Corresponding with it, ethanol produces speed also obviously faster than original strain S, and its final ethanol production is 9.33g/L, reaches 91.5% of theoretical yield.And original strain is merely 35% through the utilization to glucose about 110h, and to 140h, glucose has been consumed fully in the substratum.Final ethanol production is 7.81g/L, is 83.7% of YYJ003 bacterial strain.This demonstrates fully YYJ003 in the advantage of utilizing aspect the glucose fermentation ethanol, and its leavening property obviously is superior to original strain S.
Can find out that by Fig. 6 on the metabolic capacity to furfural, tolerance bacterial strain YYJ003 and original strain S also have very big-difference.YYJ003,, just can the furfural that add in the initial medium all be consumed, and original bacterium need about 140h through about 16h obviously faster than original strain S to the accretion rate of furfural.
6, conclusion
Tolerance bacterial strain YYJ003 can be with fast speeds metabolism fermentation inhibitor furfural; And in the substratum that has suppressor factor (concentration as stated) to exist, show fermentation character preferably; The ability of utilizing to glucose obviously is superior to original strain, and alcohol yied is higher, reaches 91.5%.
Embodiment 4
The leavening property that compares YYJ003 bacterial strain and original strain in the 10% dextrose culture-medium fermentor tank
1, test materials: with embodiment 2.
2, TP:
The preparation of seed culture medium is with embodiment 2.
The preparation of fermention medium: glucose 100g/L, yeast powder 10g/L, peptone 20g/L, furfural 1.3g/L, phenol 0.5g/L, acetate 5.3g/L, 121 ℃ of sterilization 20min.
YYJ003 bacterial strain and original strain are inoculated in respectively in the 100mL seed culture medium separately, and the YYJ003 bacterial strain is cultivated 24h at 30 ℃, 180rpm, and original strain S cultivates 12-14h under similarity condition, all with initial OD
600=1.0 are inoculated in the 3L fermention medium, under 30 ℃, 300rpm condition, cultivate, and measure thalli growth curve, glucose concn, alcohol concn and furfural concentration.
3, analytical procedure:
With embodiment 3.
4, method of calculation:
With embodiment 3.
5, test-results
By shown in Figure 7, when in fermentor tank, carrying out height sugar, high-density inoculation fermentation, compare the fermentation character that the YYJ003 performance is good with original strain.From the fermentation initial stage, thalline YYJ003 just shows stronger energy for growth, passes through lag period hardly, just can reach the logarithmic growth stage, and shows the speed of growth faster.To 17h, grow into stationary phase, fermenting process finishes basically.Original strain S is then different, in fermentor tank, through the lag period of about 45h, just shows certain growth trend, and progresses into the logarithmic growth stage.Interim at logarithmic growth, its speed of growth is still slow than YYJ003, and until about 90h, its growth progresses into stationary phase, and the highest cell density is merely about 60% of YYJ003.
Can know that by Fig. 8 YYJ003 obviously is better than original strain S to the ability of utilizing of glucose.Through about 17h, YYJ003 can be with glucose completely consumed in the nutrient solution.Corresponding with it, ethanol produces speed also obviously faster than original strain S, and its final ethanol production is 47.33g/L, reaches 92.8% of theoretical yield.And original strain need pass through about 93h could whole glucose metabolisms are intact, final ethanol production 47.25g/L is a little less than bacterial strain YYJ003.
Can find out that by Fig. 9 on the metabolic capacity to furfural, tolerance bacterial strain YYJ003 and original strain S also have very big-difference.YYJ003,, just can the furfural that add in the initial medium all be consumed, and original bacterium need about 40h through about 6h obviously faster than original strain S to the accretion rate of furfural.
6, conclusion
Tolerance bacterial strain YYJ003 can show its insensitivity to suppressor factor with fast speeds metabolism fermentation inhibitor furfural, in the substratum that has suppressor factor (concentration as stated) to exist, shows fermentation character preferably.The final ethanol production of tolerance bacterial strain is 92.8% of a theoretical value; The final ethanol production of original strain is a little less than the tolerance bacterial strain; And its fermentation time is 5.5 times of the tolerance bacterial strain, can know that relatively YYJ003 has very superior leavening property under high sugar, high-density inoculation condition.
Experiment showed, and shaking on bottle level that (content of furfural is 1.3g/L to mixed inhibitor, and the content of phenol is 0.5g/L, and the content of acetate is 5.3g/L; As it is all consistent therewith not have the specified otherwise inhibitor concentration) to low inoculum density (OD
600The growth of the YYJ003 bacterial strain=0.1) does not receive the obvious suppression effect.Final cell density reaches OD
600More than=2.0.
On the fermentor tank level, the YYJ003 bacterial strain is at low inoculum density (OD
600=0.1) in 24 hours, accomplish the substratum of fermentation include mixed suppressor factor 2% glucose under, the growth of cell is not suppressed basically.The YYJ003 bacterial strain has transformed the furfural in the substratum fully in 16 hours.Ethanol production reaches 91.5% of theoretical yield.
On the fermentor tank level, the YYJ003 bacterial strain is OD at inoculum density
600In 17 hours, accomplish the substratum of fermentation include mixed suppressor factor 10% glucose for=1.0 times, the growth of cell is not suppressed basically.The YYJ003 bacterial strain has transformed the furfural in the substratum fully in 6 hours.Its final alcohol concn is 47.33g/L, reaches 92.8% of theoretical yield.
Bacterial strain of the present invention can be grown in containing the substratum of multiple suppressor factor; The result shows that the fermentation rate of bacterial strain of the present invention improves a lot on the original basis, and the fermentation time in 2% glucose that contains mixed inhibitor and 10% dextrose culture-medium is respectively 1/7 and 1/4 of an original strain.
The YYJ003 bacterial strain contains the substratum of suppressor factor in fermentation cylinder for fermentation, and fermentation condition is: 300rpm, 30 ℃, stuffiness.Fermentation time is relevant with sugared concentration.Consisting of of substratum: glucose is 10～200g/L, and yeast powder is 1.5～20g/L, and peptone is 2～40g/L.Inoculum density is OD
600=0.1～2.0.
Claims (1)
1. the bacterial strain of the multiple suppressor factor of tolerance of a yeast saccharomyces cerevisiae, its called after yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) YYJ003 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCCNO.2757.
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| CN102296034B (en) * | 2011-08-18 | 2013-07-31 | 天津大学 | Method for obtaining yeast strain with tolerance on various inhibitors |
| CN104560750B (en) * | 2013-10-29 | 2018-01-05 | 中国石油化工股份有限公司 | A kind of method of yeast agent and application thereof and production ethanol |
| CN104560751B (en) * | 2013-10-29 | 2019-01-08 | 中国石油化工股份有限公司 | A kind of method of yeast screening assay culture medium and application thereof and screening yeast strain |
| CN105586280B (en) * | 2014-10-22 | 2019-01-25 | 天津大学 | Inositol is for enhancing purposes, the purposes of gene, expression vector of bacterial strain tolerance and application thereof, bacterial strain and application thereof |
| CN105586281B (en) * | 2014-10-22 | 2019-01-22 | 天津大学 | Proline is for enhancing purposes, the purposes of gene, expression vector of bacterial strain tolerance and application thereof, bacterial strain and application thereof |
| CN104561132B (en) * | 2015-02-15 | 2017-10-27 | 天津大学 | The method that bacterium produces ethanol is mixed in the presence of a kind of inhibitor |
| CN105462867B (en) * | 2015-11-27 | 2019-07-16 | 华南理工大学 | The saccharomyces cerevisiae of one plant of enduring high-concentration furfural and its application |
| CN105567744A (en) * | 2016-01-21 | 2016-05-11 | 天津大学 | Method for improving utilization rate of xylose in lignocellulose hydrolysate |
| CN106906152B (en) * | 2017-05-04 | 2020-06-19 | 天津大学 | Saccharomyces cerevisiae and application thereof |
| CN107937296B (en) * | 2017-11-29 | 2020-12-11 | 大连理工大学 | Recombinant saccharomyces cerevisiae with acetic acid, furfural and vanillin tolerance, and preparation method and application thereof |
| CN111100801B (en) * | 2019-09-09 | 2024-02-23 | 四川农业大学 | Furfural inhibitor tolerant yeast engineering strain, construction method and application thereof |
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| CN101434913A (en) * | 2008-12-12 | 2009-05-20 | 华东理工大学 | Wine brewing yeast strain and method for producing ethanol by efficient stalk fermentation |
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| CN101633896B (en) * | 2009-08-27 | 2011-01-05 | 中国科学院微生物研究所 | Saccharmyces cerevisiae strain for resisting high-concentration acetic acid and application thereof |
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| CN101434913A (en) * | 2008-12-12 | 2009-05-20 | 华东理工大学 | Wine brewing yeast strain and method for producing ethanol by efficient stalk fermentation |
Non-Patent Citations (2)
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