CN114317297A

CN114317297A - High-yield ethanol fermentation strain and mutation breeding method thereof

Info

Publication number: CN114317297A
Application number: CN202111342403.0A
Authority: CN
Inventors: 吴仁智; 陈东; 关妮; 陈英; 陈小玲; 曹树威; 芦志龙; 陆琦; 翁磊浩; 黄�俊; 蒙莉; 黄日波
Original assignee: Guangxi Academy of Sciences
Current assignee: Guangxi Academy of Sciences
Priority date: 2021-11-12
Filing date: 2021-11-12
Publication date: 2022-04-12
Anticipated expiration: 2041-11-12
Also published as: CN114317297B

Abstract

The invention discloses a high-yield ethanol fermentation strain with a preservation number of CCTCC No: M20211067, and a mutation breeding method of the strain comprises the following steps: selecting an original strain, placing the original strain in glycerol for storage, and preparing a culture medium for later use; inoculating and culturing in a culture medium to logarithmic phase; washing with sterile normal saline after centrifugation and controlling bacterial count at 10⁸Per mL, Co⁶⁰Irradiating to mutate the somatic cells; inoculating 15% FM culture medium for fermentation, and measuring ethanol content in the fermentation liquor to screen out a strain with high ethanol yield; continuously purifying the screened strain YPX flat plate for many times by streaking to obtain a purified high-yield strain; inoculating the high-yield strain into cane molasses, culturing and domesticating, and then carrying out strain screening and strain purification again. By the method of the invention through Co⁶⁰High-yield strains are obtained through mutagenesis, the growth speed is high, and the yield of the fermented ethanol is high.

Description

High-yield ethanol fermentation strain and mutation breeding method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a high-yield ethanol fermentation strain and a mutation breeding method thereof.

Background

Ethanol, also known as alcohol, is an important basic industrial raw material and is widely applied to a plurality of fields such as food, medicine, chemical industry, military industry and the like. There are currently three methods of ethanol production: chemical synthesis method, coal gasification method and biological fermentation method. Wherein the chemical method takes ethylene as raw material to produce ethanol by hydration; the coal chemical method is that coal is pyrolyzed to form mixed gas of CO and H2, and ethanol is produced by Fischer-Tropsch reaction; the biological fermentation method takes biomass as a raw material, and produces ethanol by fermenting and converting sugar in the biomass through microorganisms. The biological fermentation method has the advantages of mild production conditions, high product purity and the like, is widely applied in the field of medicine and food, and currently about 90 percent of ethanol in the world is produced by the fermentation method.

Bagasse is a renewable wood fiber resource, and compared with other types of wood fibers, bagasse has the advantages of centralized sources, convenience in utilization, relative cleanness and the like. According to statistics, about 1000 ten thousand tons of bagasse are produced in China every year. The components of the composite material comprise 32-48% of cellulose, 19-24% of hemicellulose, 23-32% of lignin and about 4% of ash. Because the bagasse conversion and utilization technology still has a lot of obstacles, except a few bagasse are used for papermaking, most bagasse is directly burnt out as fuel, so that the utilization rate of the total biomass is low, and the environment is polluted.

The bagasse cellulose hydrolysate is mainly glucose, the hemicellulose mainly comprises xylan, and the hydrolysate is pentose, mainly xylose and a small amount of arabinose. Therefore, the ethanol production by xylose fermentation is a way for the comprehensive utilization of the bagasse fiber at present. However, since wild-type Saccharomyces cerevisiae can not utilize xylose to produce ethanol, it is a restriction factor for the current industrialized production of cellulosic ethanol, and therefore, it is necessary to select strains capable of efficiently utilizing xylose to ferment and produce ethanol.

Disclosure of Invention

The invention aims to: aiming at the problems, the mutation breeding method of the high-yield ethanol zymophyte is provided, and the method is performed by Co⁶⁰High-yield strains are obtained through mutagenesis, the growth speed is high, and the yield of the fermented ethanol is high.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

a high-yield ethanol fermentation strain with the preservation number of CCTCC No: M20211067, the name of Pichia stipitis, the preservation number of the strain is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university, and the address is as follows: the preservation date of the eight-path Lojia mountain in Wuchang district of Wuhan city, Hubei province is 2021 year, 8 month and 23 day.

A method for fermenting ethanol by using the strain comprises the steps of selecting a culture medium, activating the strain for three times to obtain a seed solution, inoculating the seed solution into the culture medium by using the inoculation amount of 10%, and culturing at 30 ℃ and 130rpm to perform ethanol fermentation.

Further, the culture medium adopts 15% FM fermentation medium or bagasse hydrolysis concentrated solution.

A mutation breeding method of a high-yield ethanol fermentation strain comprises the following steps:

(1) early preparation: selecting an original strain, placing the original strain in glycerol for storage, and preparing a culture medium for later use;

(2) activating strains: inoculating the original strain preserved by glycerol into a culture medium, culturing for a period of time by a shaking table, then transferring for 1-3 times, and culturing under the same condition until logarithmic phase;

(3)Co⁶⁰mutagenesis: sucking bacteria liquid, centrifuging, washing with sterile normal saline for several times, collecting thallus, suspending in normal saline, and controlling bacteria number at 10⁸Per mL, taking appropriate dosage to perform Co⁶⁰Irradiating to mutate the somatic cells;

(4) strain screening: by Co⁶⁰Diluting the mutagenized bacterial liquid with normal saline, plating the diluted bacterial liquid, inoculating the diluted bacterial liquid to a plate culture medium, culturing for a proper time, selecting a single colony, inoculating the single colony to a 15% FM culture medium, fermenting, sampling and measuring the ethanol content in fermentation liquor respectively, and screening out a bacterial strain with high ethanol yield;

(5) strain purification: continuously purifying the screened strain YPX flat plate for many times by streaking to obtain a purified high-yield strain;

(6) domestication and breeding: inoculating the purified high-yield strain into cane molasses for culture and domestication, and performing the steps of strain screening and strain purification in the steps (4) and (5) again to obtain the mutagenized high-yield ethanol zymocyte.

Further, in the step (2), the shake cultivation temperature is 30 ℃, and shake cultivation is carried out at 180rpm for 12-16 h.

Further, in step (3), the centrifugation is performed at 12000rpm for 5min, and the dose is 0.4-1 KGy.

Further, in the step (4), the bacterial liquid is diluted to 10 degree^-5The culture temperature is 30 ℃, the culture time is 3d, and the fermentation is carried out for 60-72h at 30 ℃ and 130 rpm.

Further, in the step (6), the inoculation of the high-yield strain into cane molasses for culture acclimatization is to inoculate 10 ° Bx and 20 ° Bx cane molasses for culture acclimatization at 30 ℃ and 180rpm for 3-6 months.

An application of the high-yield ethanol fermentation strain in wine brewing.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. pichia stipitis is one of the most excellent strains fermented by xylose ethanol, has the advantages of high wine yield, fast growth and the like, so that the Pichia stipitis is selected for mutagenesis improvement, and Co is adopted⁶⁰The operation of irradiating and mutagenizing the pichia stipitis is simple and convenient to implement.

2. The pichia stipitis is adopted as an original strain, the growth speed is high, and after the acclimation of molasses, the fermentation performance of xylose ethanol of the strain obtained by breeding is greatly improved, the yield of ethanol is increased, and the method can be widely applied to wine brewing.

3. The xylose ethanol fermentation efficiency of the strain mutagenized by the method is 82.61%, the ethanol yield is 0.78 g/(L.h), and the yield of ethanol fermentation is 14.13% higher than that of the original strain.

4. The invention adopts cane molasses to domesticate, because molasses presents black brown, besides containing organic acid, a lot of colloid substances such as colloid, caramel and melanin, ash and other suspended substances, also containing a small amount of furan (furfural, 5-hydroxymethyl furfural), quinoline, furan, piperazine and the like, the existence of these substances is harmful to the growth of yeast, can inhibit the growth of yeast cells, influence glycolysis; molasses contains nutrient components such as trace elements, so that the bred xylose ethanol high-yield tree trunk pichia stipitis strain is domesticated by adopting cane molasses to overcome the defect.

Drawings

FIG. 1 is Co⁶⁰Comparing the primary screening results of mutagenesis;

FIG. 2 is a comparison of xylose ethanol content and relative respiration intensity;

FIG. 3 is a growth curve;

FIG. 4 is a biomass (cell dry weight) curve;

FIG. 5 is an ethanol curve;

FIG. 6 is a residual xylose curve.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to preferred embodiments. It should be noted, however, that the numerous details set forth in the description are merely for the purpose of providing the reader with a thorough understanding of one or more aspects of the present invention, which may be practiced without these specific details.

The pichia stipitis strain is selected as an original strain, the preservation number is CICC1960, the pichia stipitis strain is purchased from China center for preservation of industrial biotechnology strains, the number is 1960 for short, and the pichia stipitis strain is preserved at the ultralow temperature of-80 ℃ by adopting 25 percent (V/V) of glycerol with the final concentration.

First, preparation of culture Medium

YPX: 2% xylose, 2% peptone, 1% yeast powder, sterilizing at 121 ℃ for 20min, naturally adjusting pH, and adding 2% agar into a solid culture medium.

YPX 4: 4% xylose, 2% peptone and 1% yeast powder, sterilizing at 121 ℃ for 20min, and keeping the pH natural.

A breeding culture medium of yeast capable of growing in non-detoxified bagasse hydrolysate: adding 2% of peptone, 1% of yeast powder and sulfuric acid into the detoxified bagasse hydrolysate after acid treatment to adjust the pH value to 3.8-4.0;

fermentation of non-detoxified bagasse hydrolysate to produce ethanol culture medium: adding 15% of glucose, 2% of peptone, 1% of yeast powder and sulfuric acid into the non-detoxified bagasse hydrolysis concentrated solution to adjust the pH to 3.8-4.0. 30% glucose was added as a feed.

Manufacturers of the required components of the culture medium: peptone and yeast powder were purchased from OXOID; xylose was purchased from Sigma; the rest are Chinese medicine products.

Mutation breeding of pichia stipitis

1. Activating strains: inoculating the strain preserved in glycerol into YPX culture medium, shake culturing at 30 deg.C and 180rpm for 12-16h, transferring for 2 times, and culturing under the same conditions to logarithmic phase;

2、Co⁶⁰mutagenesis: inoculating activated 1960 strain into YPX culture medium, culturing at 30 deg.C and 180rpm to logarithmic phase, sucking bacterial liquid, centrifuging (12000rpm, 5 min), washing with sterile physiological saline for 3 times, collecting thallus, suspending in physiological saline, and controlling bacterial count to 10⁸Perml, somatic cells were mutagenized with 9 doses (0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0KGy), Co⁶⁰The irradiation being carried out at a radiation irradiation centre

3. Strain screening: by Co⁶⁰Diluting the mutagenized bacteria liquid to 10% by normal saline^-5And (3) coating the plate and inoculating a YPX plate culture medium, coating about 10 plates in each dose, culturing at 30 ℃ for 3d, selecting a single colony, inoculating a 15% FM culture medium for fermentation, culturing at 30 ℃ and 130rpm for 60-72h, respectively sampling and measuring the ethanol content in the fermentation liquor (primary screening and measuring for 48h, 60h, 72h and 80h), and screening the strain with high ethanol yield.

4. Strain purification: the yeast strains obtained by screening were continuously purified 3 times by streaking on YPX plates to obtain purified strains, which were stored at 25% (V/V) glycerol-80 ℃.

5. Domestication and breeding of cane molasses: subjecting the above to Co treatment⁶⁰Inoculating the mutagenized and bred xylose ethanol high-yield strain to 10 ° Bx and 20 ° Bx cane molasses respectively (both subjected to high-temperature sterilization treatment), culturing and domesticating at 30 ℃ and 180rpm for more than 3 months, and culturing and domesticating according to the Co⁶⁰The method for screening the strains after mutagenesis is used for screening and purifying and storing the strains.

Third, performance test

1. Preparation of bagasse hydrolysate

Drying bagasse at 60 deg.C, pulverizing, sieving with 100 mesh sieve, weighing 150g bagasse, adding 1.5L 1.5% H, and making into powder₂SO₄(W/W,%) standing at room temperature for 4h, treating with high pressure steam at 115 ℃ for 2.5h, and measuring total reducing sugar in acid-treated bagasse filtrate; the filter mud after acid treatment is added with 1.6 percent NaOH (W/V,%) for treatment for 1h at 110 ℃, and after 3 times of water washing, cellulase, beta-glucanase and xylanase are added for enzymolysis.

2. Growth Curve determination

After the activation of the strain, YPX culture was carried out at 30 ℃ and 200rpm until logarithmic phase (number of bacteria 2X 10)⁸mL), inoculated with YPX at 1% inoculum size, cultured under the same conditions, sampled at regular intervals, and assayed for OD using YPX medium as a blank₆₀₀Make 2 parallels.

3. Xylose ethanol fermentation

Adopting 15% FM fermentation medium or bagasse hydrolysis concentrated solution. After the strain is activated for three times, the seed solution is inoculated to 15 percent FM fermentation medium or bagasse hydrolysis concentrated solution with the inoculation amount of 10 percent, and is cultured in a 250mL triangular flask with the liquid loading amount of 50mL at 30 ℃ and 130rpm for ethanol fermentation. Samples were taken every 8 h. Make 2 parallels.

4. Determination of xylose content

4mL of the reaction system, measured by the DNS method, but OD was measured₅₂₀ Make 2 parallels.

5. Ethanol content determination

Gas chromatography was used to determine 2 replicates.

Fourth, test results

By using Co⁶⁰9 doses of original P.stipitis strain 1960 was mutagenized and plated to YPX plate medium. As a result, it was found that when the dose was increased to 1.2KGy, the number of colonies grown on the plate became small, and the number of colonies appeared to decrease as the dose was increased. Single colonies (10 strains per dose for a total of 90 strains) were randomly picked on plates, and the strains were deposited after three streaks. Fermenting 90 strains in xylose ethanol (in batches), wherein the fermentation conditions are as follows: samples were taken at 30 ℃ and 130rpm for 84h to determine the ethanol content. The results of primary screening mutagenesis were compared with the ethanol content of the original strain as a control, and are shown in FIG. 1. The strains can be obtained by primary screening, and the dosage is low(0.4-1KGy) a larger number of positive mutants were obtained, in particular at a 1KGy dose, and only 1 out of 10 strains selected were negative mutants. When the dosage reaches 1.2KGy, most of the obtained mutant strains are negative mutant strains, and the xylitol production of the positive mutant strains is not greatly improved compared with that of the original strain. The results for the 1.4-2KGy dose were similar to those for the 1.2KGy dose. As shown in FIG. 1, Co⁶⁰The effect of mutagenizing the pichia stipitis by adopting low dose is more ideal, and more positive mutant strains are obtained. The strain is rescreened, the strain is 1K-9 strain with better rescreening result as shown in table 1:

TABLE 1 Co⁶⁰Re-screening of mutant

The 1K-9 strain is an original strain of a high-yield strain 31.1; the 12K-1 strain is a low-yield strain 12.1; results are the mean and standard deviation of the secondary tests.

The high-producing strain 31.1, the original strain 1960 and the low-producing strain 12.1 are respectively fermented by using 15% (W/V) xylose, and the ethanol yield of 60h is shown in figure 2. The ethanol yield of the high-producing strain 31.1 is improved by 14.13 percent (highly significant, p is less than 0.001) compared with 1960, the ethanol yield of the low-producing strain 12.1 is reduced by 25.61 percent (highly significant, p is less than 0.001) compared with the original strain, and the ethanol yield of the high-producing strain 31.1 is improved by 53.42 percent (highly significant, p is less than 0.001) compared with the low-producing strain.

Three strains were inoculated with YPX, cultured at 30 ℃ and 180rpm, and the growth curves are shown in FIG. 3. The results show that the original strain grows at the highest speed, and the two mutant strains grow at similar speeds and are slower than the original strain. However, the difference among the three strains is not obvious (p is more than or equal to 0.05). In fact, the starting strain of the highly productive strain 31.1 was 1K-9, the latter having a growth rate similar to that of the original strain (no significant difference). 1K-9 is domesticated by molasses, and the fermentation performance of the xylose ethanol of the 31.1 strain obtained by breeding is improved. In addition, three strains were inoculated to 15% FM, fermented at 30 ℃ and 130rpm, and the fermentation curves are shown in FIG. 4, FIG. 5 and FIG. 6. The results show that with 15% xylose (W/V), the original strain still grew fastest, followed by the high-producing strain 31.1, as shown in fig. 4, but the difference from the original strain was not significant, the low-producing strain was slowest, which was slightly slower than the high-producing strain, the difference between the two was not significant, but the difference from the original strain was significant (p < 0.05); the high producing strain can rapidly utilize xylose to produce ethanol, the ethanol yield is the highest (figure 5), secondly, the ethanol yield is the original strain, but the difference between the two is not significant, the low producing strain is the lowest, and the ethanol content is very significant to the difference between the high producing strain (p < 0.01). The yield of xylose ethanol of the high-yield, original and low-yield strains is respectively 0.78 g/(L.h), 0.66 g/(L.h) and 0.43 g/(L.h), the corresponding conversion rate of sugar alcohol is respectively 0.3755 g/g, 0.3264g/g and 0.2887g/g, and the fermentation efficiency is respectively 81.64%, 70.96% and 62.77%; the high producing strain 31.1 had the lowest residual xylose content, followed by the original strain, but the difference between the two was not significant, and the low producing strain was the highest, which was significantly different from the high producing strain but not significantly different from the original strain (fig. 6). And after fermentation for 80 hours, the xylose utilization rates of the high-yield, original and low-yield strains are 98.52%, 97.81% and 86.83% respectively.

The results show that the fermentation performance of the high-yield strain 31.1 is superior to that of the original strain and the low-yield strain.

This example uses 9 doses of Co⁶⁰Original strains 1960 of the pichia stipitis are mutagenized, 100 colonies mutagenized with different doses are selected for xylose ethanol fermentation, a series of high-yield yeast strains and a plurality of low-yield strains are bred through primary screening, secondary screening and other ways, and the high-yield strains are domesticated for a long time by cane molasses to breed more excellent high-yield strains 31.1, wherein the ethanol yield is improved by 14.13 percent compared with the original strains.

And comparing the performances of the existing strains with those of the strains obtained in the application. Currently, the strain fermented by taking xylose as a raw material and having higher ethanol content is paecilomyces p.NF1 (ATCC-20766). The strains are respectively used for xylose fermentation of 150g/L and 200g/L, the alcohol content is respectively 7.56 percent and 9.31 percent, the conversion rate of sugar alcohol is respectively 0.39g/g and 0.38g/g, the fermentation efficiency is 75 percent, 12d and 13d are respectively needed when the fermentation period is overlong, and the ethanol yield is respectively 0.21 g/(L.h) and 0.24 g/(L.h). The high-yield strain 31.1 bred in the embodiment has the advantages that the xylose ethanol fermentation efficiency is 82.61%, the ethanol yield is 0.78 g/(L.h), and the yield is higher than that of Paecilomyces sp.NF1.

TABLE 2 comparison of fermentation Performance of xylitol ethanol

Remarking: a: average value of fermentation for 84 h; b: average value of 60h fermentation.

As can be seen from the table, although the fermentation efficiency of the high producing strain 31.1 was lower than that of the CBS 5576 strain (97.83%), the ethanol yield was higher than that of the CBS 5576 strain, which was 0.34 g/(L.h). In addition, the fermentation efficiency of the high-yield strain 31.1 is not much different from that of the inhibited two pichia stipitis strains UFMG-CM-Y2303 and UFMG-CM-Y2108, but the xylose ethanol yields of the two strains are respectively 0.39 g/(L.h) and 0.40 g/(L.h), which are obviously lower than that of the high-yield strain 31.1 (shown in a table 2). Therefore, the fermentation performance of the high-producing strain 31.1 in xylitol is in the front level in domestic similar studies.

150g of bagasse was weighed out using a 10% solid-to-liquid ratio (W/V,%), and 1.5L of 1.5% H was added₂SO₄(W/W,%) standing at room temperature for 4h, treating with high pressure steam at 115 ℃ for 2.5h, and measuring the total reducing sugar content of the acid-treated bagasse filtrate to be 33.37 +/-0.37 g/L; continuously adding 1.6 percent NaOH (W/V, percent) into the filter mud after the acid treatment, treating the filter mud for 1h at 110 ℃, washing the filter mud for 3 times, adding cellulase, beta-glucanase and xylanase, performing enzymolysis, and determining the total reducing sugar content of the bagasse hydrolysate to be 31.98 +/-0.54 g/L.

After 1% of yeast powder and 2% of peptone are added into the acid-treated non-detoxified bagasse hydrolysate, the pH is adjusted to 3.8-4.0, and a high-yield strain 31.1 and an original strain 1960 are inoculated for culture for 72 hours, so that the high-yield strain 31.1 can grow in the hydrolysate, but a control strain cannot grow. The result shows that the bred xylose ethanol high-yield strain 31.1 can grow in the non-detoxified bagasse hydrolysate.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.

Claims

1. A high-yield ethanol fermentation strain is characterized in that the preservation number is CCTCC No. M20211067.

2. A method for fermenting ethanol using the strain of claim 1, wherein: after a culture medium is selected, the strain is activated for three times to obtain a seed solution, the seed solution is inoculated into the culture medium by 10 percent of inoculation amount, and the seed solution is cultured at 30 ℃ and 130rpm for ethanol fermentation.

3. The method for fermenting ethanol by using the high-yield ethanol fermentation strain as claimed in claim 2, wherein the method comprises the following steps: the culture medium adopts 15% FM fermentation culture medium or bagasse hydrolysis concentrated solution.

4. A method of mutagenic breeding of the strain of claim 1, comprising the steps of:

(4) strain screening: by Co⁶⁰Diluting the mutagenized bacterial liquid with normal saline, plating, inoculating to plate culture medium, culturing for a proper time, selecting single colony, inoculating 15%Fermenting an FM culture medium, respectively sampling and measuring the ethanol content in fermentation liquor, and screening out a strain with high ethanol yield;

5. The mutation breeding method of high-yield ethanol fermentation bacteria according to claim 4, wherein the mutation breeding method comprises the following steps: in the step (2), the shake cultivation temperature is 30 ℃, and the shake cultivation is carried out at 180rpm for 12-16 h.

6. The mutation breeding method of high-yield ethanol fermentation bacteria according to claim 4, wherein the mutation breeding method comprises the following steps: in step (3), the centrifugation is carried out at 12000rpm for 5min, and the dose is 0.4-1 KGy.

7. The mutation breeding method of high-yield ethanol fermentation bacteria according to claim 4, wherein the mutation breeding method comprises the following steps: in the step (4), the bacterial liquid is diluted to 10^-5The culture temperature is 30 ℃, the culture time is 3d, and the fermentation is carried out for 60-72h at 30 ℃ and 130 rpm.

8. The mutation breeding method of high-yield ethanol fermentation bacteria according to claim 4, wherein the mutation breeding method comprises the following steps: in the step (6), the high-yield strain is inoculated into the cane molasses for culture and domestication, namely the high-yield strain is inoculated into the cane molasses with 10-degree Bx and 20-degree Bx, and the high-yield strain is cultured and domesticated for 3-6 months at 30 ℃ and 180 rpm.

9. Use of the high ethanol producing fermentation strain of claim 1 in brewing wine.