Saccharomyces cerevisiae IMPHA-6 and application thereof in fermentation production of ethanol

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses saccharomyces cerevisiae IMPHA-6 and application thereof in fermentation production of ethanol. The invention provides a Saccharomyces cerevisiae strain capable of resisting multiple stress, which is Saccharomyces cerevisiae IMPHA-6, and the preservation number of the Saccharomyces cerevisiae strain is CCTCC (China Center for Type Culture Collection) NO: M 20251412. The saccharomyces cerevisiae strain can solve the problem of low fermentation efficiency caused by a composite inhibitor (such as potassium chloride, ethanol, furfural and vanillin) in the lignocellulose hydrolysate, and can remarkably improve the reducing sugar utilization rate and ethanol yield during fermentation of the corn straw cellulose hydrolysate; a novel efficient microbial resource and method are provided for biomass energy production, and promotion of industrial production of cellulosic ethanol is facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a Saccharomyces cerevisiae strain IMPHA-6 and its application in fermentation production of ethanol. BACKGROUND

[0002] Lignocellulose is a renewable resource with abundant reserves, about 866.6 million tons are formed in nature every year. With the depletion of fossil energy and the aggravation of environmental problems, the use of renewable biomass resources to produce cellulosic ethanol has become an important direction of energy transformation. Corn stalks and other lignocellulosic raw materials are considered as ideal substrates for the production of second-generation biofuels due to their wide availability and low cost. However, the various inhibitors (such as furan aldehydes, phenolic compounds, organic acids and high salt ions) generated during the pretreatment and enzymatic hydrolysis of lignocellulose can severely inhibit the growth and metabolism of Saccharomyces cerevisiae, resulting in low fermentation efficiency, which is a key technical bottleneck restricting the industrial production of cellulosic ethanol.

[0003] Current solutions to the problem of hydrolysate inhibition mainly include physical and chemical detoxification treatment (such as adsorption, neutralization, evaporation) and microbial tolerance improvement. The former requires additional equipment investment and energy consumption, and is easy to cause loss of fermentable sugars; the latter mostly uses single stress factor (such as only against furfural or vanillin) laboratory adaptive evolution strategy, but the presence of multiple inhibitors in actual hydrolysate has a synergistic inhibitory effect, which makes Saccharomyces cerevisiae have a low ethanol yield in fermentation using hydrolysate and insufficient tolerance to high salt environment. In recent years, although genetically engineered strains have appeared, their genetic stability is poor and their industrial applicability is limited. Therefore, developing Saccharomyces cerevisiae strains that can simultaneously tolerate the synergistic effect of multiple inhibitors, adapt to high-concentration hydrolysate environment and have genetic stability has important industrial value for the efficient biorefinery of lignocellulosic resources.

[0004] Previous studies have shown that microorganisms can degrade lignocellulose and utilize lignocellulose for fermentation. For example, Chinese patent CN 108865914 B discloses an engineered Saccharomyces cerevisiae (INVSc-CBH2-TS) for degrading lignocellulose and its application. Chinese patent CN 119570633 A discloses a microbial complex microbial agent (including Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Fomitopsis pinicola) and a method for degrading lignocellulose. Chinese patent CN 119859587 A discloses a Saccharomyces cerevisiae (Y-6) for producing phloretin and its application. However, the above-mentioned strains do not have tolerance to lignocellulose hydrolysate inhibitors. Therefore, it is still necessary to develop new ethanol-producing strains that can utilize cellulose. Saccharomyces cerevisiae INVSc-CBH2-TS) for degrading lignocellulose and its application. Chinese patent CN 119570633 A discloses a microbial complex microbial agent (including Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Fomitopsis pinicola) and a method for degrading lignocellulose. Chinese patent CN 119859587 A discloses a Saccharomyces cerevisiae (Y-6) for producing phloretin and its application. However, the above-mentioned strains do not have tolerance to lignocellulose hydrolysate inhibitors. Therefore, it is still necessary to develop new ethanol-producing strains that can utilize cellulose. Saccharomyces cerevisiae INVSc-CBH2-TS) for degrading lignocellulose and its application. Chinese patent CN 119570633 A discloses a microbial complex microbial agent (including Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Fomitopsis pinicola) and a method for degrading lignocellulose. Chinese patent CN 119859587 A discloses a Saccharomyces cerevisiae (Y-6) for producing phloretin and its application. However, the above-mentioned strains do not have tolerance to lignocellulose hydrolysate inhibitors. Therefore, it is still necessary to develop new ethanol-producing strains that can utilize cellulose. SUMMARY

[0005] One of the objectives of this invention is to provide a strain of Saccharomyces cerevisiae that can withstand multiple stresses and improve the fermentation efficiency of lignocellulose.

[0006] This invention provides a strain of brewing yeast ( Saccharomyces cerevisiae IMPHA-6, with accession number CCTCC NO: M 20251412.

[0007] To address the shortcomings of existing brewing yeasts in fermenting lignocellulose, this invention uses the original strain S288C as the substrate strain and employs heavy ion beam irradiation combined with laboratory adaptive evolution to screen a novel brewing yeast strain, IMPHA-6. This strain exhibits multi-stress tolerance, capable of withstanding high concentrations of potassium chloride, ethanol, furfural, and vanillin individually, as well as combined stress from these substances. Its growth performance is significantly higher than that of the substrate strain. When applied to the fermentation of corn straw cellulose hydrolysate, it demonstrates ideal reducing sugar utilization and ethanol yield, solving the problem of low fermentation efficiency caused by multiple inhibitors in lignocellulose hydrolysate and providing a highly efficient microbial resource for biomass energy production.

[0008] Specifically, the strain of the present invention was obtained by the following method: using Saccharomyces cerevisiae S288C as the starting strain, after being irradiated with a carbon ion beam at a dose of 150 Gy, it was continuously passaged in a culture medium with a gradient of compound inhibitors (potassium chloride, ethanol, furfural, and vanillin), and finally the mutant strain IMPHA-6 with stable genetic traits was obtained by screening.

[0009] The brewing yeast of the present invention ( Saccharomyces cerevisiae IMPHA-6 was deposited on June 17, 2025, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China 430072, China), and classified as follows: Saccharomyces cerevisiae IMPHA-6, accession number: CCTCC NO: M 20251412.

[0010] The present invention also provides a microbial agent containing the above-mentioned brewing yeast ( Saccharomyces cerevisiae IMPHA-6.

[0011] The microbial agent of the present invention can be prepared by methods known in the art, and can be a liquid or solid microbial agent, or may include other microbial agents related to brewing yeast (Saccharomyces cerevisiae) depending on actual usage requirements. Saccharomyces cerevisiae Other strains with the same or different functions as IMPHA-6.

[0012] This invention also provides the above-mentioned brewing yeast ( Saccharomyces cerevisiae Application of IMPHA-6 or microbial agents in the fermentation production of ethanol.

[0013] In the application of this invention, the fermentation substrate for producing ethanol is corn straw cellulose hydrolysate.

[0014] The present invention also provides a method for producing ethanol by fermentation, which uses the above-mentioned brewing yeast ( Saccharomyces cerevisiae IMPHA-6 or microbial agents are used to ferment ethanol for production.

[0015] In the method of the present invention, the substrate for fermentation production is corn straw cellulose hydrolysate.

[0016] The corn stalk cellulose hydrolysate of the present invention can be prepared by methods known in the art, as long as the purpose of fully converting the cellulose and hemicellulose in lignocellulose into fermentable sugars can be achieved.

[0017] As a specific implementation method, the preparation method of the corn straw cellulose hydrolysate includes: (1) The corn stalks were pretreated with dilute sulfuric acid and then subjected to high temperature and high pressure treatment; (2) After neutralizing with alkali to neutral, wash with water, filter, and dry; (3) Enzymatic hydrolysis was performed using cellulase and xylanase. After the hydrolysis was completed, the hydrolysate was filtered and the filtrate was collected.

[0018] Preferably, the method for preparing the corn straw cellulose hydrolysate is as follows: (1) Pre-treat fresh corn stalks with dilute sulfuric acid at a concentration of 0.5% w / w; the pre-treatment time is 50-70 min; then perform high temperature and high pressure treatment at 121±2℃ and a pressure range of 100-125 kPa (15-18 psi) for 50-70 min. (2) Neutralize with sodium hydroxide, wash with water, filter, and dry; The filtration process is preferably performed using a sieve with a mesh size of 60-100 to remove salts generated by acid-base neutralization (such as Na2SO4), soluble sugars (such as arabinose), organic acids (such as acetic acid), and inhibitors (such as furfural), while retaining cellulose solids that have not been degraded by acid and some stubborn lignin (for subsequent enzymatic hydrolysis).

[0019] (3) Enzymatic hydrolysis was performed using cellulase and xylanase. After the enzymatic hydrolysis was completed, the hydrolysate was filtered and the filtrate was collected as the fermentation substrate. The preferred enzyme system is Novozymes Cellic® CTec3 HS (cellulase activity ≥250 FPU / g, xylanase activity ≥4000 U / g), with the following enzymatic hydrolysis conditions: hydrolysis temperature 50℃±2℃, pH 4.8±0.1, and hydrolysis time 5±0.5d. The hydrolysate is preferably filtered through a sieve with a mesh size of 100-200 to remove undigested lignin residue, cellulose fragments, and insoluble ash.

[0020] The final product is a cellulose hydrolysate with a theoretical yield of 75%-85% fermentable sugars (total reducing sugars).

[0021] In the method of the present invention, the fermentation production temperature is 30±2℃, the time is 48±1h, and the shaking speed is 150±10 rpm.

[0022] In the method of the present invention, the inoculum amount of the fermentation bacteria is 4-10% v / v; And / or, the culture medium for propagating the fermenting bacteria is YPD medium.

[0023] The beneficial effects of this invention are at least as follows: This invention provides a novel brewing yeast strain, IMPHA-6, which significantly enhances tolerance to single or combined stresses from potassium chloride, ethanol, furfural, and vanillin. It can also efficiently utilize corn stalk cellulose hydrolysate for ethanol fermentation. Compared with the original strain S288C, the reducing sugar utilization rate and ethanol yield are increased by 22.81% and 18.37%, respectively, showing potential for application in lignocellulose hydrolysate fermentation. Attached Figure Description

[0024] Figure 1 The results show the comparison of growth increments of Saccharomyces cerevisiae strains S288C and IMPHA-6 under potassium chloride stress. Figure 2 Comparison of growth increments between Saccharomyces cerevisiae strains S288C and IMPHA-6 under ethanol stress; Figure 3 Comparison of growth increments between Saccharomyces cerevisiae strains S288C and IMPHA-6 under furfural stress; Figure 4 Comparison of growth increments between Saccharomyces cerevisiae strains S288C and IMPHA-6 under vanillin stress; Figure 5 Comparison of the growth of Saccharomyces cerevisiae strains S288C and IMPHA-6 under combined stress conditions; Figure 6 The utilization of reducing sugars by Saccharomyces cerevisiae strains S288C and IMPHA-6 in corn straw cellulose hydrolysate; Figure 7 The fermentation of Saccharomyces cerevisiae strains S288C and IMPHA-6 in corn straw cellulose hydrolysate.

[0025] In each diagram (if any), This means P < 0.001. Detailed Implementation

[0026] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0028] The method for preparing corn stalk cellulose hydrolysate as described in the specific embodiments of this invention is as follows: Corn stalks were pretreated with dilute sulfuric acid (0.5% w / w) for 1 h, then subjected to high pressure treatment at 121℃ for 1 h (105 kPa (15 psi)). After neutralization with sodium hydroxide (pH 7.0) and subsequent washing and filtration (through an 80-mesh sieve), the corn stalk residue was dried for 48 h. Enzymatic hydrolysis was carried out using the Novozymes Cellic® CTec3 HS complex enzyme system (cellulase activity ≥250 FPU / g, xylanase activity ≥4000 U / g). The hydrolysis conditions were 50℃, pH 4.8, and 5 days. After hydrolysis, the hydrolysate was filtered (through a 150-mesh sieve), and the filtrate (cellulose hydrolysate with a theoretical yield of 80.2% fermentable sugar) was collected as the fermentation substrate.

[0029] Example 1: Screening of Saccharomyces cerevisiae mutant strains 1. Source of bacteria In this invention, the Saccharomyces cerevisiae strain S288C is used ( Saccharomyces cerevisiae S288C; Accession number: ATCC 26108; American Center for Type Culture Collection (ATCC) is the base strain. Unless otherwise specified, all chemical reagents are at least analytical grade or biological grade.

[0030] 2. Culture medium YPD liquid culture medium preparation: glucose 20 g / L, tryptone 20 g / L, yeast extract 10 g / L, sterilized at 115℃ for 30 min.

[0031] YPD solid medium preparation: glucose 20 g / L, tryptone 20 g / L, yeast extract 10 g / L and agar 20 g / L.

[0032] Stress culture medium: glucose 20 g / L, tryptone 20 g / L, yeast extract 10 g / L, with the stress reagent content added according to the specific experimental concentration.

[0033] 3. Radiation-induced mutagenesis of the strain Using the heavy ion accelerator provided by Lanzhou 12 C 6+ The original Saccharomyces cerevisiae culture was irradiated with an energy of 80 MeV / u at a dose of 150 Gy. After irradiation, the repaired culture was diluted and spread onto solid culture medium, and then screened for mutants after incubation at 30 °C for 48 h.

[0034] 4. Laboratory adaptive evolution of strains The bacterial culture to be screened was inoculated into YPD medium containing 13.4 g / L potassium chloride, 11.8 g / L ethanol, 0.3 g / L furfural, and 0.15 g / L vanillin, and the OD was aligned. 600 Then, they were transferred to YPD medium with potassium chloride concentration of 26.8 g / L, ethanol concentration of 23.6 g / L, furfural concentration of 0.6 g / L, and vanillin concentration of 0.3 g / L, and cultured for the same amount of time before being transferred to the next gradient medium, and so on, until a certain OD group was reached. 600 The value is below 0.2. At this point, the initial screening stage is complete.

[0035] The bacterial suspension obtained at the end of the initial screening stage was diluted and spread onto YPD solid medium. After incubation at 30 ℃ for 48 h, single colonies were picked and inoculated into YPD medium containing 13.4 g / L potassium chloride, 11.8 g / L ethanol, 0.3 g / L furfural, and 0.15 g / L vanillin. After incubation for a certain period of time, OD values ​​were selected. 600 Strains with an OD value higher than 0.2 were transferred to the next gradient culture medium. 600 Single colonies with a growth rate below 0.2 are eliminated, and this process continues until the growth rate of the strains slows down significantly, at which point the screening ends.

[0036] Ultimately, this invention was identified, and a strain of brewing yeast was obtained. Saccharomyces cerevisiae IMPHA-6, with accession number CCTCC NO: M 20251412, is an aerobic organism.

[0037] The above-obtained IMPHA-6 single colony culture of Saccharomyces cerevisiae was mixed with 30% glycerol at a volume ratio of 1:1 and then stored in a refrigerator at -80℃.

[0038] Example 2 Potassium Chloride Tolerance Test of Saccharomyces cerevisiae Mutant YPD medium and YPD medium containing 163.9 g / L potassium chloride stress were prepared and sterilized at 115 °C for 20 min. The above-mentioned *Saccharomyces cerevisiae* strains S288C and IMPHA-6 were cultured to the logarithmic developmental stage and aligned with OD. 600 Subsequently, 4% inoculum was added to YPD medium and stress medium, respectively, and fermentation was carried out at 30℃ and 150 rpm. OD values ​​were measured at 0 h and 48 h of culture. 600 And calculate the increment.

[0039] The results are as follows Figure 1 As shown, the mutant strain IMPHA-6 and the original strain S288C (WT) exhibited significant differences in tolerance to potassium chloride stress. In YPD medium with a potassium chloride concentration of 163.9 g / L, the mutant strain IMPHA-6 showed a significantly lower OD value after 48 h. 600 The increase was 0.336 higher than that of the original strain, representing a 240.0% improvement over the original strain.

[0040] Example 3: Ethanol Tolerance Test of Saccharomyces cerevisiae Mutant YPD medium and YPD medium containing 78.9 g / L ethanol stress were prepared and sterilized at 115 °C for 20 min. The above-mentioned *Saccharomyces cerevisiae* strains S288C and IMPHA-6 were cultured to the logarithmic growth phase and aligned with OD. 600 Subsequently, 4% inoculum was added to YPD medium and stress medium, respectively, and fermentation was carried out at 30℃ and 150 rpm. OD values ​​were measured at 0 h and 48 h of culture. 600 And calculate the increment.

[0041] The results are as follows Figure 2 As shown, the mutant strain IMPHA-6 and the original strain S288C (WT) exhibited significant differences in tolerance to ethanol stress. In YPD medium with an ethanol concentration of 78.9 g / L, the mutant strain IMPHA-6 showed a significantly lower OD value after 48 h. 600 The increase was 0.047 higher than that of the original strain S288C, representing a 15.7% improvement over the original strain.

[0042] Example 4: Furfural Tolerance Test of Saccharomyces cerevisiae Mutant YPD medium and YPD medium containing 4.0 g / L furfural stress were prepared and sterilized at 115 °C for 20 min. The above-mentioned *Saccharomyces cerevisiae* strains S288C and IMPHA-6 were cultured to the logarithmic growth phase and aligned with OD. 600 Subsequently, 4% inoculum was added to YPD medium and stress medium, respectively, and fermentation was carried out at 30℃ and 150 rpm. OD values ​​were measured at 0 h and 48 h of culture. 600 And calculate the increment.

[0043] The results are as follows Figure 3 As shown, the mutant strain IMPHA-6 and the original strain S288C (WT) exhibited significant differences in tolerance to furfural stress. In YPD medium with a furfural concentration of 4.0 g / L, the mutant strain IMPHA-6 showed a significantly lower OD value after 48 h. 600 The increase was 0.137 higher than that of the original strain S288C; it was 148.9% higher than that of the original strain.

[0044] Chinese patent CN 107937296 A discloses that the recombinant Saccharomyces cerevisiae S288C-YPL162C, overexpressing the YPL162C gene, showed significantly better growth than S288C-HO in a fermentation medium containing 3 g / L furfural, with a maximum cell density 52% higher than the original strain. However, its maximum tolerated concentration was still lower than the maximum furfural tolerated concentration of 4.0 g / L for the mutant strain IMPHA-6 of this invention. Chinese patent CN 105462867 A discloses a Saccharomyces cerevisiae strain CGMCC No. 11356 tolerant to high concentrations of furfural. This strain showed significantly better growth and fermentation capacity than the control strain in a simulated lignocellulose hydrolysate containing 2.0 g / L furfural, but its maximum tolerated concentration was still lower than the maximum furfural tolerated concentration of 4.0 g / L for the mutant strain IMPHA-6 of this invention.

[0045] Example 5: Vanillin tolerance test of Saccharomyces cerevisiae mutant strain Prepare YPD medium and YPD medium containing 2.2 g / L vanillin stress, and sterilize at 115 ℃ for 20 min. Culture the above-mentioned *Saccharomyces cerevisiae* S288C and IMPHA-6 strains to the logarithmic growth phase and OD aligned. 600 Subsequently, 4% inoculum was added to YPD medium and stress medium, respectively, and fermentation was carried out at 30℃ and 150 rpm. OD values ​​were measured at 0 h and 48 h of culture. 600 And calculate the increment.

[0046] The results are as follows Figure 4 As shown, the mutant strain IMPHA-6 and the original strain S288C (WT) exhibited significant differences in tolerance to vanillin stress. In YPD medium with a vanillin concentration of 2.2 g / L, the mutant strain IMPHA-6 showed a significantly lower OD value after 48 h. 600 The increase was 0.375 higher than that of the original strain S288C, representing an improvement of 183.8% over the original strain.

[0047] Chinese patent CN 107937296 A has disclosed that the recombinant Saccharomyces cerevisiae S288C-YPL162C overexpressing the YPL162C gene showed significantly better growth than S288C-HO in a fermentation medium containing 2 g / L vanillin, with the maximum cell density increasing by 71.4% compared to the control strain. However, it was still lower than the maximum tolerated concentration of vanillin 2.2 g / L of the mutant strain IMPHA-6 of this invention.

[0048] Example 6: Test on the growth of a Saccharomyces cerevisiae mutant under combined stress YPD liquid medium and YPD medium containing a compound stress of 53.6 g / L potassium chloride, 47.3 g / L ethanol, 1.2 g / L furfural, and 0.6 g / L vanillin were prepared and sterilized at 115 °C for 20 min. The above-mentioned *Saccharomyces cerevisiae* strains S288C and IMPHA-6 were cultured to the logarithmic developmental stage and aligned with the OD. 600 Subsequently, the culture medium was inoculated at a rate of 4% and cultured at 30 °C and 150 r / min. OD was measured every 24 h. 600 A total of 72 hours of testing were conducted.

[0049] The results are as follows Figure 5 As shown, under combined stress, the mutant strain IMPHA-6 exhibited significantly better growth than the original strain S288C (WT). After 72 h of culture, the OD of the mutant strain IMPHA-6 was significantly higher. 600 It was 0.168 higher than the original strain, representing a 22.5% improvement.

[0050] Chinese patent CN 106906152 A discloses a new strain of Saccharomyces cerevisiae, E7-12, which is resistant to multiple inhibitors and high temperatures. Its growth and fermentation capacity in a compound inhibitor medium containing furfural 0.39 g / L, phenol 0.15 g / L and acetic acid 1.59 g / L are significantly better than those of xylose-utilizing strain E7. However, its tolerance concentration of the compound inhibitor is still significantly lower than that of the mutant strain IMPHA-6.

[0051] Example 7: Testing of reducing sugar utilization rate of a *Saccharomyces cerevisiae* mutant strain in corn straw cellulose hydrolysate. Single colonies of the above-mentioned Saccharomyces cerevisiae S288C and IMPHA-6 strains were picked and cultured in YPD medium to the logarithmic phase, and the OD was aligned. 600 The inoculum was then introduced into the corn straw cellulose hydrolysate at an inoculum rate of 8% (v / v). After fermentation at 30 ℃ and 150 rpm for 48 h, the reducing sugar in the fermentation broth was determined by the DNS method.

[0052] The specific testing method is as follows: Dissolve 1 g DNS and 20 g alkaline sodium tartrate in distilled water, and add distilled water to 100 mL to obtain DNS reagent. Prepare a series of glucose standard solutions with known concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.0 mg / mL), and take 1 mL of each standard solution into a test tube. Take 1 mL of fermentation hydrolysate solution sample into a test tube, and add 1 mL of DNS reagent to each standard solution and sample test tube, and mix well. Place the test tube in a boiling water bath and heat for 5-10 min until the solution color turns orange-red. Place the test tube in an ice bath and quickly cool to room temperature. Use a spectrophotometer to measure the absorbance of each standard solution and sample at a wavelength of 540 nm. Plot a standard curve with glucose concentration on the x-axis and absorbance on the y-axis. Calculate the corresponding reducing sugar concentration from the standard curve based on the absorbance of the sample.

[0053] The results are as follows Figure 6 As shown, both strains can ferment in corn straw cellulose hydrolysate, and the mutant strain IMPHA-6 has a better reducing sugar utilization capacity than the original strain. After 48 h of fermentation, the remaining reducing sugar of the mutant strain IMPHA-6 was only 14.51 g / L, while that of the original strain was 17.82 g / L. The reducing sugar utilization rate of the mutant strain IMPHA-6 in corn straw cellulose hydrolysate was 22.81% higher than that of the original strain.

[0054] Example 8: Ethanol Yield Test of Saccharomyces cerevisiae Mutant in Corn Stalk Cellulose Hydrolysate Single colonies of the above-mentioned Saccharomyces cerevisiae S288C and IMPHA-6 strains were picked and cultured in YPD medium to the logarithmic phase, and the OD was aligned. 600 Then, the inoculum was added to the corn straw cellulose hydrolysate at an inoculum rate of 8% (v / v). After fermentation at 30 ℃ and 150 rpm for 48 h, 1 ml of the fermentation broth was taken, diluted 10 times, and the ethanol content in the fermentation broth was detected using an SBA-40D biosensor analyzer.

[0055] The results are as follows Figure 7 As shown, both strains can ferment in corn straw cellulose hydrolysate, and the mutant strain IMPHA-6 has a better fermentation capacity than the original strain. After 48 h of fermentation, the ethanol yield of mutant strain IMPHA-6 was 25.64 g / L, while that of the original strain was only 21.66 g / L. The ethanol yield of mutant strain IMPHA-6 in corn straw cellulose hydrolysate was 18.37% higher than that of the original strain.

[0056] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A strain of brewer's yeast ( Saccharomyces cerevisiae IMPHA-6, characterized in that, The accession number is CCTCC NO: M 20251412.

2. A microbial agent, characterized in that, Contains the brewing yeast as described in claim 1 ( Saccharomyces cerevisiae IMPHA-6.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent can be a liquid bacterial agent or a solid bacterial agent.

4. The brewing yeast as described in claim 1 ( Saccharomyces cerevisiae The application of IMPHA-6 or the microbial agent according to claim 2 or 3 in the fermentation production of ethanol.

5. The application according to claim 4, characterized in that, The fermentation substrate for producing ethanol is corn straw cellulose hydrolysate.

6. A method for producing ethanol by fermentation, characterized in that, The brewing yeast as described in claim 1 ( Saccharomyces cerevisiae IMPHA-6 or the microbial agent described in claim 2 or 3 is used to produce ethanol through fermentation.

7. The method according to claim 6, characterized in that, The substrate for the fermentation process is corn straw cellulose hydrolysate.

8. The method according to claim 7, characterized in that, The preparation method of the corn straw cellulose hydrolysate includes: (1) The corn stalks were pretreated with dilute sulfuric acid and then subjected to high temperature and high pressure treatment; (2) After neutralizing with alkali to neutral, wash with water, filter, and dry; (3) Enzymatic hydrolysis was performed using cellulase and xylanase. After the hydrolysis was completed, the hydrolysate was filtered and the filtrate was collected.

9. The method according to any one of claims 6-8, characterized in that, The fermentation process was carried out at a temperature of 30±2℃ for 48±1h and a shaking speed of 150±10 rpm.

10. The method according to any one of claims 6-9, characterized in that, The inoculation amount of the fermentation bacteria is 4-10% v / v; And / or, the culture medium for propagating the fermenting bacteria is YPD medium.

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