CN104480030A - High-temperature-resistant Spathaspora passalidarum mutant strain and application of strain in producing ethanol by fermenting xylose - Google Patents
High-temperature-resistant Spathaspora passalidarum mutant strain and application of strain in producing ethanol by fermenting xylose Download PDFInfo
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Abstract
The invention discloses a high-temperature-resistant Spathaspora passalidarum mutant strain and an application of the strain in producing ethanol by fermenting xylose, belonging to the technical field of microbial fermentation. In order to solve the problem that common industrial fermenting microorganisms such as saccharomyces cerevisiae cannot produce ethanol by using xylose fermentation, the invention provides a novel yeast strain-Spathaspora passalidarum U-30. The strain is preserved in CGMCC on 28th, October, 2014 with the preservation number of CGMCC No: 9863. The strain is obtained by ultraviolet mutagenesis of an original strain. The yield of ethanol fermented by a shaking flask at 37 DEG C is improved by 36.5% compared with that of the original strain, the temperature tolerance is improved to 41 DEG C and the ethanol tolerance is improved to 12%.
Description
Technical field:
The invention belongs to technical field of microbial fermentation, be specifically related to the resistant to elevated temperatures yeast Spathaspora passalidarumU-30 that a strain obtains through Uv-induced screening and the method utilizing xylose production ethanol thereof.
Background technology:
Along with petroleum-based energy be on the verge of exhaust, and threaten grain security with the first-generation bio-ethanol that the farm crop such as corn are raw material, utilize from agricultural, industry, the lignocellulose biomass in forestry and urban waste is the concern that the s-generation bio-ethanol of raw material production is more and more subject to each big country.Lignocellulose is renewable resources the abundantest on the earth.Utilizing lignocellulosic material to produce alcohol fuel is a kind of effective ways solving energy dilemma, crisis of resource and environmental pollution.In lignocellulosic material, Mierocrystalline cellulose, hemicellulose, xylogen proportion are about 4:3:3, and wherein the main hydrolysate of hemicellulose is wood sugar, are second largest carbohydrates in lignocellulosic material.The productive rate of the ethanol obtained by xylose determines the transformation efficiency that lignocellulosic material is converted into ethanol.
Xylose fermentation for producing ethanol can not be utilized for general industrial fermentation microorganism such as yeast saccharomyces cerevisiae.Natural wood-sugar fermentation yeast is as pichia stipitis, although can effective xylose fermentation ethanol, when utilizing Production of Alcohol from Lignocellulose, bacterial strain can suppress by the compound that produces of pretreated lignocellulosic material.The more important thing is, pichia stipitis is when muscovado ferments, and the existence of glucose can suppress the utilization of wood sugar.Spathaspora passalidarum is the saccharomyces neoformans that a strain finds naturally to utilize wood-sugar fermentation recently.Research finds, utilizes the speed of wood sugar higher than the speed utilizing glucose, can utilize the glucose in ligno-cellulose hydrolysate, wood sugar, cellobiose mixed fermentation, and the existence of glucose does not have restraining effect to wood-sugar fermentation under oxygen restricted condition.
Thermophilic fermentation is significant for alcohol production, improves leavening temperature and can reduce apparatus cools energy consumption, reduce cooling water amount, thus effectively reduce alcohol production cost.Meanwhile, for the technique that saccharification and fermentation are synchronously carried out, require to improve leavening temperature, make it to mate with saccharification temperature, the bacterial strain with thermophilic fermentation ability has more industrial application potentiality as far as possible.
Summary of the invention:
The object of the present invention is to provide a strain resistant to elevated temperatures Spathaspora passalidarum mutant strain and utilize wood-sugar fermentation to produce the method for ethanol.
With Spathaspora passalidarum NRRL Y-27907 for starting strain carries out ultraviolet mutagenesis, test tube is utilized to carry out primary dcreening operation after mutagenesis, shake flask fermentation carries out multiple sieve, obtains the mutant strain U-30 that a strain ethanol tolerance and temperature tolerance are all better than original strain.
Described Spathaspora passalidarum mutant strain is that starting strain obtains after Uv-induced screening by Spathaspora passalidarum NRRLY-27907, be specially Spathaspora passalidarumU-30, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 28th, 2014 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCC No:9863.
The ethanol production of described bacterial strain shake flask fermentation at 37 DEG C improves 36.5% than original strain, and its temperature tolerance brings up to 41 DEG C, and alcohol resistance brings up to 12%.
Described bacterial strain optimum growth temperature is 28 DEG C, and pH is 5.5.
The method of described strain fermentation wood sugar producing and ethanol is as follows:
(1) acquisition of seed liquor: choose a ring bacterium from inclined-plane to YPD liquid nutrient medium, 30 DEG C, 180rpm cultivates 12h is seed liquor for subsequent use according to being cultured to the logarithmic growth middle and later periods under inoculum size the same terms of 1% from first cell culture medium.
(2) shake flask fermentation is cultivated or ferment tank cultivation:
Fermention medium: wood sugar 60 ~ 100g/L, corn steep liquor 15 ~ 25g/L, potassium primary phosphate 2.5g/L, magnesium sulfate heptahydrate 0.1g/L, pH4.5 ~ 5.5.
Conditions of flask fermentation: inoculum size 5 ~ 10%, temperature 30 DEG C, shaking speed 120 ~ 150rpm, liquid amount 80 ~ 120/250mL.
Ferment tank condition: inoculum size 5 ~ 10%, temperature 30 DEG C, rotating speed 120 ~ 300rpm, liquid amount 50 ~ 80%, air flow 0.02 ~ 0.05v/vmin.
Beneficial effect:
1 compares with original strain, and in the test of Du Shi pipe, former bacterium is at 40 DEG C of substantially no longer aerogenesis, and U-30 is at 40 DEG C of aerogenesis speed, still can aerogenesis under 41 DEG C of conditions, and visible U-30 is stronger than former bacterium heat-resisting ability.Former bacterium can aerogenesis when alcohol concn is 10%, but when alcohol concn is 12% just no longer fermentation gas; U-30, still can aerogenesis when alcohol concn reaches 12%.Visible, U-30 is stronger than the alcohol resistance of former bacterium.
2, mutant strain U-30 under the high temperature conditions ethanol fermentation performance significantly improve, at 37 DEG C, the ethanol production of shake flask fermentation is 16.45g/L, improves 36.5% than original strain; Mutant strain at 30 DEG C, conditions of flask fermentation bottom fermentation 5 days, alcohol concn reaches 39.04g/L, alcohol getting rate is 0.39g/g, and xylose utilization rate is 100%, and throughput rate is 0.325g/Lh, and the alcohol getting rate of former bacterium is 0.30g/g, compares with former bacterium and improve 30%; Mutant strain is at 30 DEG C, and in 5L fermentor tank, fermentation time reduction is 72h, xylose utilization rate 100%, alcohol concn reaches 39.875g/L, and alcohol getting rate is 0.40g/g, reach 87% of theoretical yield, throughput rate reaches 0.553g/Lh, and the alcohol getting rate of former bacterium is 0.32g/g.Visible, no matter mutant strain is utilize the performance in xylose fermentation for producing ethanol process to be all better than original strain at high temperature, low temperature, shaking flask or fermentor tank.
Accompanying drawing illustrates:
The wood sugar consumption in 5L fermentor tank of Fig. 1 U-30 and former bacterium;
Fig. 2 U-30 and the ethanol production of former bacterium in 5L fermentor tank.
Embodiment:
The present invention is described below by specific embodiment.Unless stated otherwise, technique means used in the present invention is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the material component in these embodiments and consumption or change also belong to protection scope of the present invention.
Embodiment 1:Spathaspora passalidarumU-30 mutant strain is screened
1. with Spathaspora passalidarum NRRL Y-27907 for starting strain, under ultraviolet condition, 20W, irradiate 20s, get the bacterium liquid 1mL after mutagenesis transfer in xylose concentration be 2% YPX liquid nutrient medium, transfer in fresh 2%YPX liquid nutrient medium after 40 DEG C of lucifuges cultivate 24h, transfer in fresh 2%YPX liquid nutrient medium after 24h again, until OD
620value no longer changes.Be spread evenly across on xylose plate after getting the dilution of this bacterium liquid, picking mutant strain after 30 DEG C of lucifuges cultivation 1-2d.
The YPX substratum of 2%: wood sugar 20g/L, peptone 20g/L, yeast leaching powder 10g/L, pH nature.
Xylose plate: wood sugar 20g/L, peptone 20g/L, yeast leaching powder 10g/L, agar 2%, pH nature.
2. the mutant strain obtained in pair 1 carries out the fermentation of xylose media test tube, 30 DEG C, and cultivate 5d under 180r/min, centrifugal, DNS method measures the content of wood sugar in supernatant liquor, just sifts out the mutant strain that xylose utilization energy force rate starting strain is strong.
3. the mutant strain obtained in pairs 2 carries out shake flask fermentation, and fermention medium is: 100g/L wood sugar, 20g/L peptone, 10g/L yeast leaching powder, pH nature.Fermentation condition is: liquid amount 100mL, inoculum size 5%, 37 DEG C, and 100rpm ferments 5 days.Centrifugal, the content of ethanol in high-performance liquid chromatogram determination supernatant liquor, sifts out ethanol high-yield bacterial strain U-30 again.Ethanol production comparatively starting strain has and significantly promotes, and fermentation results is in table 1.
Table 1 mutant strain U-30 compares with starting strain ethanol growing amount
Wood sugar shake flask fermentation producing and ethanol is utilized at embodiment 2:30 DEG C
Contrast each other with U-30 and original strain, carry out ethanol fermentation experiment
(1) acquisition of seed liquor: respectively choose a ring bacterium to 50mLYPD liquid nutrient medium from inclined-plane, 30 DEG C, 180rpm cultivates 12h, from first cell culture medium according to the inoculum size of 1% in the 250mL shaking flask of 50mLYPD liquid nutrient medium, being cultured to the logarithmic growth middle and later periods under the same terms is seed for subsequent use.
(2) fermention medium moiety: wood sugar 100g/L, corn steep liquor 15g/L, dipotassium hydrogen phosphate 2.5g/L, magnesium sulfate heptahydrate 0.1g/L, pH5.5.
(3) fermentation condition: inoculum size is 7%, during fermentation, shaking speed is 120rpm, and during fermentation, liquid amount is 100mL.
(4) ferment 5 days, get supernatant and utilize high-efficient liquid phase technique to detect wood sugar in fermented liquid and alcohol concn.
Result shows, after U-30 ferments 5 days, alcohol concn reaches 39.04g/L, alcohol getting rate is 0.39g/g, xylose utilization rate is 100%, and throughput rate is 0.325g/Lh, and former bacterium ferment 5 days after alcohol concn be 30.43g/L, wood sugar residue 18.72g/L, alcohol getting rate is 0.30g/g, compares with former bacterium, and the alcohol getting rate of U-30 improves 30%.
Embodiment 3:30 DEG C of 5L ferment tank producing and ethanol
Contrast each other with U-30 and original strain, utilize 5L fermentor tank enlarged culturing to carry out ethanol fermentation experiment.
Fermention medium is: wood sugar 100g/L, corn steep liquor 15g/L, dipotassium hydrogen phosphate 2.5g/L, magnesium sulfate heptahydrate 0.1g/L, pH5.5.
Fermentation condition is: inoculum size 7%, temperature 30 DEG C, rotating speed 150rpm, air flow 0.1L/min.
Result, as accompanying drawing 1, Fig. 2, can be found out, in 5L fermentor tank, the fermentation time of U-30 obviously shortens than in shaking flask, and fermentation period is 72h, and wood sugar has all utilized, and ethanol production also reaches 39.857g/L, reaches 86.6% of theoretical yield.Former bacterium still has part sugar not utilize completely to 84h, and ethanol production also only reaches 32.73g/L, and continue to extend fermentation time wood sugar and substantially do not consume, alcohol concn declines on the contrary to some extent.
Embodiment 4: alcohol resistance compares
(1) be linked in 5mLYEPD liquid tube respectively by former bacterium and U-30,30 DEG C, 180rpm overnight incubation, as seed liquor for subsequent use.
(2) be placed with in the sterile test tube of Du Shi pipe add various composition according to the requirement in table 1 respectively in inversion, get 1mL seed liquor in test tube, mixing of vibrating gently.Quiescent culture at 30 DEG C, every 24h observes aerogenesis and record, the results are shown in Table 2.
Table 1
| Ethanol concn (%, v/v) | 2 | 4 | 6 | 8 | 10 | 12 |
| 15 ° of dense wheat juice (ml) of Brix | 4 | 4 | 4 | 4 | 4 | 4 |
| Dehydrated alcohol (ml) | 0.2 | 0.4 | 0.6 | 0.8 | 1 | 1.2 |
| Water (ml) | 4.8 | 4.6 | 4.4 | 4.2 | 4 | 3.8 |
| Seed (ml) | 1 | 1 | 1 | 1 | 1 | 1 |
Table 2
Note: "-" represents not aerogenesis, and "+" represents aerogenesis, the gas production rate how many expressions of "+" are different, " ++++" represent that bubble is full of.
As can be seen from Table 2, former bacterium can fermentation gas when alcohol concn is 10%, but when alcohol concn is 12% just no longer fermentation gas; U-30, still can aerogenesis when alcohol concn reaches 12%.Visible, U-30 is stronger than the alcohol resistance of former bacterium.
Embodiment 5: temperature tolerance compares
(1) be linked in 5mLYEPD liquid tube respectively by former bacterium and U-30,30 DEG C, 180rpm overnight incubation, as seed liquor for subsequent use.
(2) in the sterile test tube of Du Shi pipe of putting upside down, 4mL15 ° of dense wheat juice of Brix, 5mL sterilized water and 1mL seed liquor is added respectively in test tube, to vibrate gently mixing, quiescent culture at 30 DEG C, 35 DEG C, 37 DEG C, 39 DEG C, 40 DEG C, 41 DEG C respectively, every 12h observes aerogenesis and record, the results are shown in Table 3.
Table 3
Note: "-" represents not aerogenesis, and "+" represents aerogenesis, the gas production rate how many expressions of "+" are different, " ++++" represent that bubble is full of.
As can be seen from Table 3, former bacterium is at 40 DEG C of substantially no longer aerogenesis, and U-30 is at 40 DEG C of aerogenesis speed, still can fermentation gas under 41 DEG C of conditions.Visible U-30 is stronger than former bacterium temperature tolerance.
Claims (5)
1. a strain thermotolerant yeast mutant strain, is characterized in that, described mutant strain is specially Spathaspora passalidarum U-30, and deposit number is CGMCC No:9863.
2. a strain thermotolerant yeast mutant strain as claimed in claim 1, it is characterized in that, described mutant strain can tolerate the ethanol of 41 DEG C of high temperature and 12%.
3. utilize wood-sugar fermentation to produce a method for ethanol, it is characterized in that, use the high temperature resistant mutant strain described in claim 1 to ferment for producing bacterial strain, concrete steps are as follows:
(1) seed culture: carry out two-stage in YPD substratum and cultivate to obtain seed liquor for subsequent use;
(2) fermentation culture:
Conditions of flask fermentation: inoculum size 5 ~ 10%, temperature 30 DEG C, shaking speed 120 ~ 150rpm, liquid amount 80 ~ 120/250mL.
Ferment tank condition: inoculum size 5 ~ 10%, temperature 30 DEG C, rotating speed 120 ~ 300rpm, liquid amount 50 ~ 80%, air flow 0.02 ~ 0.05v/vmin.
4. a kind ofly as claimed in claim 3 utilize wood-sugar fermentation to produce the method for ethanol, it is characterized in that, the substratum of described fermentation culture is: wood sugar 60 ~ 100g/L, corn steep liquor 15 ~ 25g/L, potassium primary phosphate 2.5g/L, magnesium sulfate heptahydrate 0.1g/L, pH4.5 ~ 5.5.
5. a strain thermotolerant yeast mutant strain as claimed in claim 1 produces the application in ethanol in wood-sugar fermentation.
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| CN105505804A (en) * | 2016-01-15 | 2016-04-20 | 天津科技大学 | Mutant strain capable of efficiently fermenting xylose and method of using mutant strain for fermentation to produce ethanol |
| CN114874928A (en) * | 2022-05-02 | 2022-08-09 | 大连理工大学 | Method for improving stress resistance of cellulose ethanol production strain through heat shock protein overexpression |
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| CN101845404B (en) * | 2010-01-08 | 2013-05-08 | 广西科学院 | Brewing yeast strain, breeding method thereof, and application of the strain in alcohol production |
| CN103232948B (en) * | 2013-05-10 | 2015-07-15 | 天津科技大学 | High-temperature resistant saccharomyces cerevisiae strain and breeding method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105505804A (en) * | 2016-01-15 | 2016-04-20 | 天津科技大学 | Mutant strain capable of efficiently fermenting xylose and method of using mutant strain for fermentation to produce ethanol |
| CN105505804B (en) * | 2016-01-15 | 2019-06-14 | 天津科技大学 | One plant height imitates the mutant strain of xylose-fermenting and the method using its producing and ethanol that ferments |
| CN114874928A (en) * | 2022-05-02 | 2022-08-09 | 大连理工大学 | Method for improving stress resistance of cellulose ethanol production strain through heat shock protein overexpression |
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