C6/C5 CO-FERMENTED SACCHAROMYCES CEREVISIAE CAPABLE OF RELIEVING ANTAGONISM BETWEEN HIGH XYLOSE UTILIZATION AND HIGH ROBUSTNESS AND APPLICATION THEREOF

Abstract

The present invention further discloses an application of Saccharomyces cerevisiae in the fermentation of a second generation fuel, ethanol, with a straw lignocellulose biomass hydrolysate as a raw material. Experiments prove that the strain of the prevent invention has an efficient co-fermentation capacity of C6/C5 while tolerating multiple inhibitors, and can consume all the glucose and xylose to produce ethanol; and the sugar-alcohol conversion rate is up to 0.43 g g.sup.−1. The strain and screening strategy used in the present invention provide technical reference and basis for the further breeding of a Saccharomyces cerevisiae strain which has a superior fermenting property and is produced by the second generation fuel, ethanol.

Claims

1. A C6/C5 co-fermented Saccharomyces cerevisiae capable of relieving antagonism between high xylose utilization and high robustness, characterized in that the strain simultaneously has the properties of high inhibitor robustness and high xylose utilization, and named Saccharomycescerevisiae 6M-15; and the strain has been deposited on Aug. 17, 2020 at “China General Microbiological Culture Collection Center” (CGMCC) under the accession number: CGMCC No. 20436.

2. An application of the C6/C5 co-fermented Saccharomyces cerevisiae capable of relieving antagonism between high xylose utilization and high robustness of claim 1 in the fermentation of a second generation fuel, ethanol, with a straw lignocellulose biomass hydrolysate as a raw material.

3. The application of claim 2, characterized in that the method for the fermentation production of a second generation fuel, ethanol with a straw lignocellulose biomass hydrolysate as a raw material is as follows: wherein a medium having a formula of 20 g L.sup.−1 peptone and 10 g L.sup.−1 yeast powder is named a YP medium; water in the YP medium is replaced with a cornstalk hydrolysate, such that the fermentation medium has the following concentrations of components: 20 g L.sup.−1 peptone, 10 g yeast powder, 50.23±0.39 g L−1 glucose, 25.62±0.47 g xylose, 0.93±0.13 g L.sup.−1 cellobiose, 1.03±0.31 g L.sup.−1 galactose, 8.09±0.24 g arabinose, 4.36±0.28 g L.sup.−1 acetic acid, 1.09±0.25 g 5-hydroxymethyl furfural, 0.35±0.06 g L.sup.−1 furfural, and 4.78±0.13 g L.sup.−1 total phenol compounds, pH=3.5±0.2; and wherein the fermentation conditions are as follows: a culture temperature is 30±2° C., a charging ratio is subjected to charging a 40 mL medium in a 150 mL oxygen-limited flask, a shaker speed is 200±20 rpm; a rubber plug is used for sealing, a syringe needle is inserted to control the oxygen-limited conditions; an initial inoculation OD value is 10; all the glucose and xylose are completely consumed after culturing for 96±10 h under oxygen-limited conditions; a fermentation product is centrifuged and filtered to obtain a supernatant containing ethanol.

4. The application of claim 3, characterized in that the method for the fermentation production of a second generation fuel, ethanol with a straw lignocellulose biomass hydrolysate as a raw material is as follows: wherein a medium having a formula of 20 g L.sup.−1 peptone and 10 g L.sup.−1 yeast powder is named a YP medium; water in the YP medium is replaced with a cornstalk hydrolysate, such that the fermentation medium has the following concentrations of components: 20 g L.sup.−1 peptone, 10 g yeast powder, 50 g L−1 glucose, 25 g L.sup.−1 xylose, 0.93 g L.sup.−1 cellobiose, 1.03 g L.sup.−1 galactose, 8.09 g arabinose, 4.36 g L.sup.−1 acetic acid, 1.09 g L.sup.−1 5-hydroxymethyl furfural, 0.35 g furfural, and 4.78 g L.sup.−1 total phenol compounds, pH=3.5±0.1; and wherein the fermentation conditions are as follows: a culture temperature is 30° C., a charging ratio is subjected to charging a 40 mL medium in a 150 mL oxygen-limited flask, a shaker speed is 200 rpm; a rubber plug is used for sealing, a syringe needle is inserted to control the oxygen-limited conditions; an initial inoculation OD value is 10; all the glucose and xylose are completely consumed after culturing for 96±1 h under oxygen-limited conditions; a fermentation product is centrifuged and filtered to obtain a supernatant containing ethanol.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 shows growth situation of an original strain LF1 on a YP medium of a high-toxicity cornstalk pretreating fluid.

[0027] FIG. 2 shows fatality rate of the original strain LF1 mutagenized by ARTP.

[0028] FIG. 3 shows a high-resistant mutant strain screened by Bioscreen.

[0029] FIG. 4 shows oxygen-limited fermenting properties of different strains on a YP medium containing 50% (v/v) pretreating fluid and 40 g L.sup.−1 YPX medium; (a) denotes growth of LF1 and LF1-6 on a YP medium containing 50% (v/v) pretreating fluid; (b) denotes growth of LF1 and LF1-6 on a YPX medium (40 g L.sup.−1 xylose); (c) denotes ethanol consumption; and (d) denotes ethanol yield.

[0030] FIG. 5 shows oxygen-limited fermenting properties of the original strain LF1 and mutant strains LF1-6M and 6M-15 on the YPX medium (40 g xylose); (a) denotes strain growth; (b) denotes xylose metabolism; and (c) denotes ethanol yield.

[0031] FIG. 6 shows oxygen-limited fermenting properties of the original strain LF1(a) and mutant strain 6M-15(b) on high-concentration mixed sugars (80 g L.sup.−1 glucose and 40 g xylose).

[0032] FIG. 7 shows oxygen-limited fermenting properties of the original strain LF1(a) and mutant strain 6M-15(b) on a YP medium of a non-detoxification cornstalk hydrolysate.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The present invention will be further described in detail with reference to the specific drawings and examples. The examples below are merely preferred embodiments of the present invention. It should be indicated that the description below is merely for explaining the present invention, but not for limiting the present invention in any form. Any simple amendment, equivalent alteration and modification made to the embodiments on the basis of the technical spirit of the present invention shall fall within the scope of the technical solution of the present invention.

[0034] Unless otherwise specified, the materials, reagents and the like used in the examples are available commercially.

Example 1 Determination of Microbiological Media and Basic Culture Conditions

[0035] (1) Saccharomyces cerevisiae Medium

[0036] YP medium: 20 g L.sup.−1 peptone and 10 g L.sup.−1 yeast powder; YPD medium: 20 g L.sup.−1 glucose was added to the YP medium; YPX medium: 40 g xylose was added to the YP medium; and YPGX medium: 40 g xylose and 80 g L.sup.−1 glucose were added to the YP medium. YP medium containing pretreating fluid: different proportions of cornstalk pretreating fluid were added according to experiment demands; full-hydrolysate YP medium: water in the YP medium was replaced with hydrolysate. 20 g g agar powder was added to the corresponding solid medium.

[0037] (2) Preparation of a Cornstalk Pretreating Fluid and Hydrolysate

[0038] Absolutely dry cornstalk was crushed into particles having a diameter of less than 1 mm, and 100 g straw particles were mixed with 0.5% dilute sulphuric acid according to a solid-to-liquid ratio of 1:5, then transferred to a stainless steel tube reactor, and heated up to 180° C. for reacting for 40 min, and the reaction mixture was centrifuged to obtain a supernatant, namely, the supernatant was the cornstalk pretreating fluid (hereinafter referred to as pretreating fluid).

[0039] The reaction mixture was transferred to a shake flask, and NaOH served to adjust pH to 4.8, 20 FPU/g cellulase was added for enzymolysis, where reaction temperature was 50° C., revolving speed was 200 rpm, enzymolysis time was 48 h; and the supernatant obtained by centrifugation was namely, the cornstalk hydrolysate (hereinafter referred to as hydrolysate).

[0040] Major sugar components of the pretreating fluid and hydrolysate, and concentration of the inhibitor were shown in Table 1.

[0041] Table 1 List of the components of the cornstalk pretreating fluid and hydrolysate

[0042] (3) Basic Culture Conditions

[0043] Culture conditions of oxygen-limited shake flask: culture temperature was 30° C.; 150 mL oxygen-limited flask was loaded with a 40 mL medium, shaker speed was 200 rpm and the shake flask was sealed by a rubber plug; a syringe needle was inserted to control the oxygen-limited conditions, three parallel fermentation experiments were performed, and sampling was performed for once per 4 h.

[0044] Culture conditions of the Bioscreen full-automatic growth curve analyzer: culture volume was 200 μL, initial inoculum size OD 600 was 0.2; culture temperature was 30° C., three parallel fermentation experiments were performed, and automatic sampling was performed to detect OD values per 0.5 h.

Example 2 Determination on the Detection Method of Raw Components and Fermentation Products

[0045] The concentrations of the pretreating fluid, hydrolysate as well as substrate and product of ethanol during fermentation were measured by high performance liquid chromatography.

[0046] 1 mL sample was taken and centrifuged at a high speed (13000 r/min, 5 min) to remove impurities or bacteria in the sample; supernatant was taken and filtered by a 0.22 μm microfiltration membrane; and the component content thereof was measured by a high performance liquid chromatography system Waters e2695. Standard sample concentration: 49.998 g L.sup.−1 glucose, 48.891 g L.sup.−1 xylose, 9.857 g L.sup.−1 cellobiose, 6.029 g L.sup.−1 galactose, 6.074 g L.sup.−1 arabinose, 0.630 g L.sup.−1 5-hydroxymethyl furfural, and 1.042 g L.sup.−1 furfural. Chromatographic conditions: {circle around (1)} glucose, xylose, arabinose, cellobiose, galactose, acetic acid and ethanol were analyzed by an HPX-87H ion-exchange chromatography (Bio-Rad Aminex); 5 mM H.sub.2SO.sub.4 served as a mobile phase of the chromatographic column at 45° C., and a Waters 2414R1 differential refraction detector was used; {circle around (2)} furfural and 5-HMF were detected by a Waters 2998PDA UV detector and WondaSil C18 chromatographic column (GL Sciences) at 40° C. with 40% methanol as a mobile phase. The content of total phenols in the pretreating fluid was detected by a folin reagent; vanillic aldehyde served as a standard substance and was reacted with a Folin-Ciocalteu reagent to detect its absorption value at 725 nm.

Example 3 Breeding of a C6/C5 Co-Fermented Saccharomyces cerevisiae Capable of Relieving Antagonism Between High Xylose Utilization and High Robustness Strain

[0047] The original strain is a C6/C5 co-fermented Saccharomyces cerevisiae strain LF1 constructed by at an earlier stage of the applicant's laboratory; and the strain has been applied for a patent with the application number: 201510747241.7 and accession number: CGMCC No. 11331. The C6/C5 Co-fermented Saccharomyces cerevisiae capable of relieving antagonism between high xylose utilization and high robustness strain was expected to be obtained by iterative mutagenesis at atmospheric pressure and room temperature plasma (ARTP) and in combination with alternative domestication and screening in a high-toxicity (containing a plurality of inhibitors) cornstalk pretreating fluid and a pure xylose medium; and the specific technical steps were as follows:

[0048] (1) Determination on the Concentration of a High-Toxicity Cornstalk Pretreating Fluid Significantly Inhibiting Growth of an Original Strain

[0049] Influences of the adding amount of the pretreating fluid in the medium on the growth of the original strain were tested. Saccharomyces cerevisiae YP media were respectively added 0% (control), 10%, 30%, and 50% (v/v) pretreating fluid; the original strain LF1 had an initial inoculation OD 600 of 0.2, an inoculation volume of 40 mL, culture temperature of 30° C., revolving speed of 200 rpm and culture time of 24 h. Results were shown in FIG. 1; when the pretreating fluid had an adding proportion of 50% (v/v), the strain growth was obviously inhibited. Based on this, the adding amount of the 50% (v/v) pretreating fluid could be determined as a pressure condition to screen a strain having improved inhibitor robustness in the follow-up experiments.

[0050] (2) ARTP Mutagenisis

[0051] {circle around (1)} Test on the influences of different radiation time on the fatality rate of the original strain

[0052] Single colonies of the original strain were picked and cultured to the mid-log phase, and centrifuged to collect bacteria solution, washed by sterile water for 2-3 times; a proper amount of solution was diluted to a bacterial suspension having an OD 600 value of 0.6-0.8; 10 μL bacterial suspension were taken and coated on a sterile metal slide glass; 4 time gradients, 0 s (control), 15 s, 30 s, and 45 s were configured in an ARTP mutation breeding apparatus, and 3 parallel samples were set to each gradient for mutagenisis. After all the samples were treated, slide glasses were respectively put to EP tubes loaded with 1 mL sterile water with sterile forceps, and oscillated for 5 min by a vibrator; then cells attached on the slide glass were eluted to sterile water to form a new bacterial suspension; then the new bacterial suspension was concentrated to 100 μL and completely coated on aYPD plate, cultured at 30° C.; after growing bacterial colonies, fatality rate was calculated according to the quantity variance of the bacterial colonies between the experimental group and control group.

[0053] {circle around (2)} Determination of mutagenesis conditions After being irradiated for 15 s at 120 W power and

[0054] 10 SLM gas flow, the original strain had a fatality rate of 97.2%; and when irradiated for 30 s, no strain survived (as shown in FIG. 2). Therefore, 15 s mutagenesis time, 120 W power and 10 SLM gas flow served as conditions of ARTP mutation breeding.

[0055] {circle around (3)} Based on the mutagenesis conditions determined in {circle around (2)}, the mutagenized original strain was put on a YPD medium and incubated for 30 min for recovery growth. Finally, the bacterial cells were put on a YP medium containing 50% (v/v) pretreating fluid for domesticated culture. The doubling of cell biomass served as a transfer indicator; after through the transferred incubation for domesticated culture for about 30 d, doubling time of the cell biomass significantly shortened and kept the same, cells were coated on a YP solid medium containing 50% (v/v) pretreating fluid to separate single colonies.

[0056] (3) Screening of the Strain Having Significantly Improved Robustness

[0057] Cells in (2)® were coated on a YP solid medium containing 50% (v/v) pretreating fluid for culture at 30° C.; after bacterial colonies grew, bacterial colonies having a relatively large diameter were picked; and a Bioscreen full-automatic growth curve analyzer (Oy GrowthCurves Ab Ltd, Helsinki, Finland) was used to test the growth performance of the mutant strains on the YP medium containing 50% (v/v) pretreating fluid (as shown in FIG. 3); the culture volume was 200 μL; initial inoculum size OD 600 was 0.2; and culture temperature was 30° C. Strains growing faster were screened again under a same condition of a 40 mL medium to obtain a strain having the highest robustness, named LF1-6.

[0058] Batch fermentation test was performed on the 50% (v/v) pretreating fluid medium; and fermentation conditions were as follows: culture temperature was 30° C.; 150 mL oxygen-limited flask was loaded with a 40 mL 50% (v/v) pretreating fluid medium; shaker speed was 200 rpm and the flask was sealed with a rubber plug; then a syringe needle was inserted to control the oxygen-limited conditions; initial inoculum size OD was 0.2; sampling was performed per 4 h; the fermentation experiment was repeated for three times; and a mean value of the data was taken for calculation. Results (as shown in FIG. 4a) showed that LF1-6 had significantly enhanced robustness, while almost no original strain grew. But on the 40 g L.sup.−1 xylose medium, the xylose utilization rate (as shown in FIG. 4c) and ethanol generation rate (as shown in FIG. 4d) of the mutant strain LF1-6 were obviously lower than these of the original strain LF1; and there was an antagonism relationship between high xylose utilization and high robustness. The mutant strain LF1-6 was alternatively domesticated on the 50% (v/v) pretreating fluid medium and the pure xylose medium to screen a strain having improved xylose metabolic capability, named LF1-6M; and the strain was fermented for 20 h on the same xylose medium; the OD 600 improved 44.23%, xylose utilization rate improved 6.58%. But compared with the xylose utilization rate of the original strain LF1, there was still a big difference (42.4% vs 95.6%); the antagonism problem could be not solved effectively (as shown in FIGS. 5a and b).

[0059] (4) Screening of a Strain Relieving Antagonism Between Xylose Utilization and Robustness by Multi-Round Iterative ARTP Mutagenesis and Anaerobic Culture

[0060] Directed to the above antagonism problem, LF1-6M, as an original strain, was subjected to multi-round iterative ARTP mutagenesis, and anaerobic culture on a pure xylose solid YPX medium; and alternatively domesticated on a YP medium containing 50% (v/v) pretreating fluid and YPX medium; the growth OD 600 value served as a detection indicator; when the strain was domesticated till the cell biomass doubled, the time significantly shortened and kept the same. A proper amount of the final bacterial suspension was coated on a YP medium containing 50% (v/v) pretreating fluid; after bacterial colonies grew, bacterial colonies having a larger diameter were picked to test the xylose utilization capacity and robustness; thus finally screening a mutant strain, namely, a strain having synchronously improved robustness and xylose metabolism, named Saccharomyces cerevisiae 6M-15; and the strain has been deposited on Aug. 17, 2020 at “China General Microbiological Culture Collection Center” (CGMCC) (address: No. 3 of No. 1 Yard, West Beichen Road, Chaoyang District, Beijing) under the accession number: CGMCC No. 20436.

Example 4 Comparison of the Fermenting Property and Ethanol Yield of Different Mutant Strains Between YPX and YPGX Media

[0061] (1) Media: YPX medium: 20 g L.sup.−1 peptone, 10 g L.sup.−1 yeast powder, and 40 g L.sup.−1 xylose; YPGX medium: 20 g L.sup.−1 peptone, 10 g L.sup.−1 yeast powder, 80 g L.sup.−1 glucose, and 40 g xylose.

[0062] (2) Activation of strains: a single colony of a single strain to be tested was picked and inoculated on a 5 mL YPX fluid medium for culture for 24 h at 30° C. and 200 rpm; then transferred on a 10 mL YPX fluid medium for secondary activation for 12 h. Activated strains were respectively inoculated on 150 mL conical flasks containing 40 mL YPX and YPGX fluid media for fermentation culture.

[0063] (3) Fermentation conditions: culture temperature was 30° C.; 150 mL oxygen-limited flask was loaded with a 40 mL medium, shaker speed was 200 rpm and the flask was sealed by a rubber plug; a syringe needle was inserted to control the oxygen-limited conditions; initial inoculation OD value was 0.2, sampling was performed for once per 4 h to detect the OD value; and the sample was centrifuged and filtered to obtain a supernatant; the HPLC method in Example 2 was taken to detect the change of the sugar components and fermentation products; three parallel experiments were performed for each strain.

[0064] The fermentation results showed as follows:

[0065] On a pure xylose YPX medium, the mutant strain had gradually improved xylose utilization capacity (as shown in FIG. 5), LF1≈6M-15>LF1-6M; 6M-15 could completely utilize xylose when fermented for 24 h, basically being up to the xylose metabolic level of the original strain LF1; and the sugar-alcohol conversion rate at the peak of ethanol yield was up to 90.98% of the theoretical value. Further, 6M-15 showed excellent fermenting properties (as shown in FIG. 6) on a mixed YPGX medium containing 80 g L.sup.−1 glucose and 40 g L.sup.−1 xylose. 6M-15 could completely consume the sugar components within 28 h; compared with the original strain, the fermentation time shortened 8 h (28 h vs36 h); and the maximum ethanol concentration might be up to 54.98 g L.sup.−1; and the sugar-alcohol conversion rate might be up to 91.37% of the theoretical value. It can be seen from the above results that the high-robustness strain had a recovered xylose metabolic capability through multi-round ARTP iterative mutagenesis and anaerobic screening.

Example 5 Ethanol Fermenting Properties of the Original Strain LF1 and Mutant Strain 6M-15 on a Cornstalk Hydrolysate YP Medium

[0066] (1) Cornstalk hydrolysate YP medium: cornstalk hydrolysate was used to substitute the water in the YP medium (20 g L.sup.−1 peptone and 10 g L.sup.−1 yeast powder) Major sugar components and concentrations of the medium: 50 g L.sup.−1 glucose and 25 g L.sup.−1 xylose; as well as major inhibitors and concentrations: 4.36 g L.sup.−1 acetic acid, 1.09 g 5-hydroxymethyl furfural, 0.35 g L.sup.−1 furfural and 4.78 g L.sup.−1 total phenol compounds, pH=3.5.

[0067] (2) The reactivation process of the strains was set forth in Example 4.

[0068] (3) Fermentation conditions were set forth in Example 4; initial inoculation OD value was 10; sampling was performed for once per 12 h; and the detection method was set forth in Example 2.

[0069] The fermentation results showed as follows:

[0070] In the fermentation process of a full hydrolysate YP medium having a low pH (about 3.5) and containing more higher concentration of inhibitors, the growth of the original strain LF1 was seriously inhibited; the glucose metabolic rate was slow; almost no xylose was utilized; at the end of the fermentation, LF1 only utilized 50% glucose, and only produced about 7.5 g ethanol. However, the growth of the mutant strain 6M-15 was almost not influenced; the glucose utilization rate was faster, all the glucose could be utilized within 24 h while utilizing xylose synchronously; at the end of the fermentation, 6M-15 might completely utilize glucose and xylose; ethanol yield was 30.53 g L.sup.−1; sugar-alcohol conversion rate was 0.43 g L.sup.−1, being up to 85% of the theoretical value (as shown in FIG. 7).

[0071] When concentrations of acetic acid, 5-hydroxymethyl furfural, furfural and total phenol compounds and other major inhibitors in the straw biological hydrolysate were not higher than the above concentration, and pH was not lower than 3.5, the strain had obvious growth vigor; and the ethanol yield produced through the transformation of glucose and xylose was not lower than 0.43 g.sup.−1.

[0072] It can be seen from the above results that the mutant strain 6M-15 finally screened in the present invention simultaneously has the characteristics of high xylose utilization rate and high robustness to inhibitors in hydrolysate, thus significantly relieving the prominent antagonistic problem between high xylose utilization and high robustness in the breeding process of a Saccharomyces cerevisiae strain with lignocellulose biomass hydrolysate as a raw material to produce a second generation fuel, ethanol. To sum up, the breeding 6M-15 strain (accession number: CGMCC No. 20436) in the present invention has the ability of resisting multiple inhibitors in lignocellulose biomass hydrolysate while possessing a C6/C5 co-fermentation capacity. The strain is a potential strain applied to the industrial production of a second generation fuel, ethanol. Moreover, the strain provides excellent substrate cells for the further breeding of the Saccharomyces cerevisiae strain which has more excellent fermenting property and produced by the second generation fuel, ethanol.

Example 6 Application of the C6/C5 Co-Fermented Saccharomyces cerevisiae Capable of Relieving Antagonism Between High Xylose Utilization and High Robustness in the Fermentation of a Second Generation Fuel, Ethanol, with a Straw Lignocellulose Biomass Hydrolysate as a Raw Material

[0073] The method for shake-flask fermentation of a second generation fuel, ethanol with a straw lignocellulose biomass hydrolysate as a raw material was as follows:

[0074] water in a YP medium was replaced with a cornstalk hydrolysate, such that the fermentation medium had the following concentrations of components: 20 g L.sup.−1 peptone, 10 g L.sup.−1 yeast powder, 50 g L−1 glucose, 25 g L.sup.−1 xylose, 0.93 L.sup.−1 cellobiose, 1.03 g L.sup.−1 galactose, 8.09 g arabinose, 4.36 g L.sup.−1 acetic acid, 1.09 g L.sup.−1 5-hydroxymethyl furfural, 0.35 g L.sup.−1 furfural, and 4.78 g L.sup.−1 total phenol compounds, pH=3.5±0.1; and the fermentation conditions were as follows: a culture temperature was 30° C., a charging ratio was subjected to charging a 40 mL medium in a 150 mL oxygen-limiting flask, a shaker speed was 200 rpm; a rubber plug was used for sealing, a syringe needle was inserted to control the oxygen-limited conditions; an initial inoculation OD value was 10; all the glucose and xylose were completely consumed after culturing for 96±1 h under oxygen-limited conditions; a fermentation product was centrifuged and filtered to obtain a supernatant containing ethanol.

[0075] Specific operating steps were set forth in Examples 4 and 5.