CN103789396B - Method for rapidly screening high-yield ethanol saccharomycetes - Google Patents

Abstract

A method for quickly screening high-ethanol-producing yeasts. First, the growth curve of the strain is drawn, and the lag phase, logarithmic growth phase, and stable phase are judged accordingly; several test tube liquid culture media are prepared and distributed, and half of them are placed upside down and filled with For the fermentation small tube of the liquid medium, sterilize the two kinds of test tube culture medium by high pressure steam, take it out and cool it; insert the strains into the liquid medium of the test tube without the fermentation small tube, shake the culture to the end of logarithmic growth, and take it out for use; During the bacterial culture period, prepare the antibiotic mother solution, filter and sterilize; aseptically remove the fermentation tubule from the test tube liquid culture medium, put it upside down into the above-mentioned culture solution that has been cultivated to the end of logarithmic growth, and ensure that there are no air bubbles in the tubule. Add an appropriate amount of antibiotic solution, and turn it to static fermentation culture; after a period of culture, take out the above test tube liquid, observe the size of the air column in the fermentation tube, the larger the air column, the higher the ethanol production, so that the target can be quickly screened out. strain.

Description

A method for rapid screening of high ethanol-producing yeast

technical field

The invention belongs to the technical field of microbiology, and relates to a rapid preliminary screening method for high-yielding microorganism strains, especially a rapid screening method for microorganisms that can utilize glucose to produce ethanol under anaerobic conditions.

Background technique

Strain screening has always been one of the important research contents in the field of microbiology, and it is also one of the difficulties that need to be solved in related scientific research work. For directed screening of microorganisms, such as directed mutagenesis, the screening of target mutant strains is complex and important, and if there is no screening marker, the screening of target strains will be more blind and burdensome. With regard to the screening methods of the target strains, researchers at home and abroad have done a lot of research and discussion, and also put forward many different ideas. These ideas ease some research tasks, and the purpose and direction are clearer, but the workload is still huge. Therefore, it is necessary to further explore the screening method of the target strain.

On the other hand, when the energy crisis forces human beings to develop and utilize new energy sources, the selection of yeast strains with high ethanol production has become one of the key influencing factors for converting cellulose or hemicellulose to ethanol.

For yeast, the metabolic mechanism of glucose utilization is as follows:

In a medium that uses glucose as a carbon source (such as YEPD medium) or a medium that can be converted to glucose (such as PDA medium), yeast such as Saccharomyces cerevisiae can metabolize glucose under both aerobic and anaerobic conditions . When there is oxygen in the environment, the yeast can completely oxidize part of the glucose to obtain the energy needed for growth and reproduction, and at the same time produce CO ₂ ; Ethanol and CO ₂ are produced.

With oxygen: C ₆ H ₁₂ O ₆ +6O ₂ 6CO ₂ +6H ₂ O+energy

Without oxygen: C ₆ H ₁₂ O ₆ 2CO ₂ +2C ₂ H ₅ OH+energy (small amount)

Due to this characteristic of yeast metabolism, it was proposed to use fermentation (Dunchen) tubules to qualitatively indicate the ability of yeast to produce CO ₂ , thereby indirectly indicating the ability of yeast to produce ethanol. However, a disadvantage of this is that the yeast performs aerobic metabolism in the early stage of cultivation, that is, during the growth and reproduction period, completely oxidizes glucose to obtain energy, and produces CO ₂ at the same time, and the efficiency of producing CO ₂ is relatively high. One molecule of glucose is completely oxidized It can generate 6 molecules of CO ₂ ; while in anaerobic metabolism, 1 molecule of glucose can only be converted into 2 molecules of CO ₂ . In this way, it is not easy to distinguish whether the CO ₂ in the fermentation tube is produced through aerobic metabolism or anaerobic metabolism, or what is the ratio of the former to the latter. If these CO ₂ are regarded as glucose fermentation without distinction , it is easy to get false high-yielding strains.

It should be noted that when the growth conditions change, the yeast will choose a certain metabolic method. For example, under aerobic conditions, yeast mainly uses aerobic metabolism. At this time, most of the consumed glucose is metabolized in aerobic form, but a very small part is consumed in ethanol fermentation; while under anaerobic conditions, Yeast mainly uses anaerobic metabolism. At this time, most of the consumed glucose is metabolized in the form of anaerobic fermentation, but a small part is still consumed in the form of aerobic metabolism. Therefore, aerobic metabolism and anaerobic fermentation cannot be strictly distinguished during the entire culture cycle of yeast. Nevertheless, we can still use the metabolic characteristics of yeast under anaerobic conditions to screen target strains.

Contents of the invention

The object of the present invention is to provide a method for quickly and accurately screening yeast strains with high ethanol production in view of the above-mentioned problems. The present invention utilizes the characteristic that yeast mainly ferments ethanol under anaerobic conditions to design a test scheme.

Technical scheme of the present invention is:

A method for rapidly screening high ethanol-producing yeast, the steps of the method are:

Step 1, first draw the growth curve of the bacterial strain, and judge its lag phase, logarithmic growth phase, and stable phase accordingly;

Step 2, preparing and distributing liquid culture medium in several test tubes, putting half of them upside down into a small fermentation tube filled with liquid medium, autoclaving the two test tube culture media, taking them out and cooling them;

Step 3: Insert the strains into the test tube liquid culture medium without the fermentation tube, shake the culture until the end of logarithmic growth, and take it out for use;

Step 4, during bacterial culture, prepare antibiotic mother solution, filter and sterilize;

Step 5, aseptic operation Take out the fermentation tube in step 2 from the test tube liquid culture medium, put it upside down into the culture medium cultivated in the above step 3 to the end of logarithmic growth, ensure that there are no air bubbles in the tube, and add Appropriate amount of antibiotic solution prepared in the 4th step is transferred to static fermentation culture;

Step 6. After culturing for one cycle, take out the test tube liquid in the above step 5, and observe the size of the air column in the fermentation tube. The larger the air column, the higher the ethanol production, so that the target strain can be quickly screened out.

The concrete operation of the inventive method is as follows:

(1) Drawing of growth curve of yeast strain

Under the optimal culture conditions determined in previous experiments, including culture medium composition and content, culture temperature, pH, culture cycle, shaking speed, inoculate yeast in liquid medium and culture, and the culture time is regarded as horizontal. Coordinates, taking the absorbance value _A600 of the bacterium liquid as the ordinate, draw the growth curve of the absorbance value _A600 of the yeast culture solution changing with the culture time, and judge the lag period, logarithmic growth period, and stable period of bacterial strain growth accordingly;

(2) Preparation and sterilization of test tube liquid medium

Prepare glucose-containing medium, such as YEPD or a medium that can be converted into glucose, such as PDA medium or bean sprout juice glucose medium or wort glucose medium 1000mL, and divide into 100 test tubes, each 9.7mL, take which 50 test tube liquids, put them upside down into a fermentation tube filled with 0.3mL of the above liquid medium, carefully operate to avoid air bubbles in the small tube, record the test tube liquid medium with the fermentation tube as a, and the other 50 untreated The liquid culture medium in the test tube containing the fermentation small tube is denoted as b, and at the same time, it is sterilized at 112°C and 0.5Mpa humid heat steam for 20min, and then taken out and cooled to room temperature;

(3) Inoculation and cultivation

Inoculate the yeast strain into the test tube liquid medium b without a fermentation tube with an inoculum amount of 0.5%, and culture it with shaking at 150 r/min at 30°C until the end of the logarithmic growth phase, take it out for use, and record it as c;

(4) Preparation of antibiotic mother solution

Prepare kanamycin solution mother liquor and chloramphenicol solution mother liquor respectively, concentration is 0.5g/100mL, two kinds of antibiotic mother liquors are filtered and sterilized under aseptic operation respectively; The preparation of antibiotic mother liquor is carried out in the bacteria liquid cultivation stage after inoculation;

(5) Ethanol fermentation

Under aseptic operation, take out the fermentation tube in a from the liquid in the test tube, quickly put it upside down and put it into the culture medium c cultivated to the end of the logarithmic growth phase, operate carefully so that no air bubbles enter the fermentation tube, and then rapidly transfer to the same tube Add kanamycin solution with a final concentration of 50 μg/mL and chloramphenicol solution with a final concentration of 25 μg/mL to the culture solution. Static culture to one culture cycle;

A culture cycle includes a cell growth stage that does not produce ethanol and a cell fermentation glucose to produce ethanol and CO ₂ stage;

(6) Primary screening of ethanol-producing strains

Take out the above-mentioned culture solution c that has completed one culture cycle, observe the bubble generation in the fermentation tubule and the discharge of liquid in the test tube liquid, and judge the amount of ethanol produced by the yeast according to the size of the gas column in the fermentation tubule. The larger it is, the higher the ethanol production is indirectly, so that the target strain with high ethanol production can be screened out very quickly; the amount of ethanol production is determined by further quantitative detection;

(7) Verification of preliminary screening results

The high-yielding strains obtained from the above primary screening were subjected to ethanol-producing fermentation, and then samples were taken, diluted appropriately, and analyzed for ethanol production by using a biosensing analyzer or liquid chromatography to verify the accuracy of the primary screening results of this method.

Advantages and beneficial effects of the present invention :

According to the different metabolic characteristics of yeast under aerobic and anaerobic conditions, the present invention rationally arranges the time for placing the fermentation tubules, skillfully eliminates the interference of its _CO2 production under aerobic conditions on the fermentation judgment, and ensures the _CO2 in the fermentation tubules Most of the gas is produced by ethanologenic fermentation rather than aerobic metabolism. This makes it more accurate to judge the ethanol-producing ability of the yeast according to the number of air bubbles in the small fermentation tube. In addition, the method is not only suitable for the screening of general ethanol-producing yeasts, but is especially suitable for the screening of yeast mutagenic strains. This program is logically rigorous, simple and easy to operate, and is a fast and accurate screening method.

Description of drawings

Figure 1 is the growth curve of Saccharomyces cerevisiae QD in YEPD.

Figure 2 is the growth curve of S. pombe 2.1621 in YEPD.

Figure 3 is the growth curve of Kluyveromyces marxense 191l in YEPD.

Figure 4 is the growth curve of Saccharomyces cerevisiae QD in PDA.

Figure 5 is a growth curve of S. pombe 2.1621 in PDA.

Figure 6 is the growth curve of Kluyveromyces marxense 191l in PDA.

Figure 7 is the growth curve of Saccharomyces cerevisiae QD in bean sprout juice glucose medium.

Fig. 8 is the growth curve of S. pombe 2.1621 in bean sprout juice glucose medium.

Fig. 9 is the growth curve of Kluyveromyces marx 191l in the bean sprout juice glucose medium.

Detailed ways

Example 1: Preliminary judgment on the ability of yeast to ferment glucose to produce ethanol in YEPD medium

The yeast strains capable of fermenting glucose to produce ethanol are Saccharomyces cerevisiae QD, derived from Tsingtao Brewery Co., Ltd., and Schizosaccharomyces pombe 2.1621, derived from the Culture Collection Center of the Chinese Academy of Kluyveromyces marxianus 191l was obtained from the China Industrial Microbiology Collection Center, and all three were published strains.

According to the conventional experimental method, it is necessary to test the ethanol-producing ability of each yeast in the experiment.

The concentration of viable bacteria in the inoculum solution is 1.0×10 ⁶ ~ 1.0×10 ⁷ CFU/mL; the yeast YEPD medium solution is an aqueous solution composed of the following components, and the contents of each component are yeast in g/1000mL aqueous solution powder 10.0, peptone 20.0, glucose 20.0, the balance is water, and the pH value of the culture medium is natural; the sterilization method of the culture medium is high-pressure steam sterilization, and the sterilization temperature is 112°C for 20 minutes.

The best culture conditions for each strain have been determined through previous experiments as culture temperature 30°C, culture cycle 24h, shaking rate 150r/min, and natural pH.

(1) Draw the growth curve of yeast

Under the optimal culture conditions determined in the previous experiment, including the content of each component of the YEPD medium, culture temperature, culture cycle, pH, and shaking rate, the yeast was inoculated in the liquid medium and cultured. is the abscissa, and the absorbance value A ₆₀₀ of the bacterial solution is the ordinate, samples are taken every 2 hours, the absorbance value A ₆₀₀ of the solution is measured, and the growth curve of the absorbance value A ₆₀₀ of each yeast culture solution is drawn with the culture time. To determine the lag phase, logarithmic growth phase, and stable phase of each strain, refer to the accompanying drawings, wherein accompanying drawing 1 is the QD growth curve of Saccharomyces cerevisiae, accompanying drawing 2 is the growth curve of Schizosaccharomyces pombe 2.1621, and accompanying drawing 3 is the Max Kluyveromyces 191l growth curve.

(2) Preparation and sterilization of test tube liquid medium

Prepare 1000mL of glucose-containing YEPD medium, and divide it into 100 test tube liquids, each 9.7mL; take 50 of the test tube liquids, put them into an inverted fermentation tube filled with 0.3mL of the above liquid medium, and carefully operate to Make sure that there are no air bubbles in the small tube, record the liquid culture medium in the test tube with the fermentation small tube as a, and the liquid culture medium in the other 50 test tubes without the fermentation small tube, mark it as b; Sterilize for 20 minutes, take out and cool.

(3) Inoculation and cultivation

Inoculate the yeast liquid into the test tube liquid medium without the fermentation tube with an inoculation amount of 0.5%, and shake and culture at 150 r/min at 30°C for 8 hours. , denoted as c.

(4) Preparation of antibiotic mother solution

Prepare kanamycin solution mother solution and chloramphenicol solution mother solution respectively, the concentration is 0.5g/100mL, two kinds of antibiotic mother solutions are filtered and sterilized respectively under aseptic operation;

(5) Ethanol fermentation

Under aseptic operation, take out the fermentation tube in a, quickly put it upside down and put it into the culture solution c that has been cultivated to the end of the logarithmic growth phase, operate carefully so that no air bubbles enter the fermentation tube, and then quickly add to the culture solution respectively Kanamycin solution with a final concentration of 50 μg/mL and chloramphenicol solution with a final concentration of 25 μg/mL. After the above operations are completed, continue to culture the test tube culture c with the fermentation tube at 30°C until one Cultivation cycle.

A culture cycle includes the thalline growth stage without putting the fermentation tubule and the thalline fermentation glucose production ethanol and _CO2 stage after being placed in the fermentation tubule.

(6) Preliminary screening of ethanol high-yielding strains

Take out the above-mentioned culture solution c that has completed one culture cycle, observe the bubble generation in the fermentation tubule and the discharge of liquid in the test tube liquid, and judge the amount of ethanol produced by the yeast according to the size of the air column in the fermentation tubule. Larger, the more ethanol is produced, so that strains with high ethanol production can be screened out very quickly, and the amount of ethanol produced can be determined by further quantitative detection such as liquid chromatography or biosensor analyzer.

(7) Verification of preliminary screening results

The high-yielding strains obtained from the above primary screening were subjected to ethanol-producing fermentation, samples were taken, diluted appropriately, and ethanol production was analyzed using the SBA-40C biosensor analyzer to verify the correctness of this scheme.

For the growth curve of yeast, please refer to attached drawing 1, attached drawing 2, and attached drawing 3. Since the nutrients in the YEPD medium are rich and easy to use, the yeast will soon enter the logarithmic growth phase after a short delay period. According to the growth curve: The lag phase of Saccharomyces cerevisiae QD growth is 0-2h, the logarithmic growth phase is 2-10h, and enters the stable phase after 10h; the growth lag phase of S. pombe 2.1621 is 0-2h, and the logarithmic growth phase is 2- After 10h and 10h, it enters the stable phase; the growth delay phase of Kluyveromyces marx 191l is 0-2h, the logarithmic growth phase is 2-10h, and it enters the stable phase after 10h of cultivation;

Compare the size of the air column in the fermentation tubules of each strain, and see the attached table 1 for the results

Note: According to the amount of gas produced in the fermentation tube, it is divided into 5 grades:

a "++++" indicates that the fermentation tube is filled with gas,

b "+++" means that the fermentation tube is filled with 3/4 gas,

c "++" means that the fermentation tube is filled with 1/2 gas,

d "+" indicates that the fermentation tube is filled with 1/4 gas,

e "-" indicates that there is no gas in the fermentation tube

Verification of preliminary screening results

Carry out ethanol-producing fermentation analysis on the gas-producing strains listed in the table above to verify the correctness of the preliminary screening results. The concentration of the prepared ethanol standard solution was 40mg/100mL, and after the fermentation liquid was diluted 40 times, the ethanol content of each strain was measured as shown in Table 2

Note: The ethanol production value in the above table is measured after the fermentation stock solution is diluted 40 times

It can be seen from the data in Table 2 that the ethanol yield of Saccharomyces cerevisiae QD is relatively high, which verifies the preliminary screening results listed in Table 1, indicating that the experimental protocol is correct.

Example 2: Preliminary judgment on the ethanol production capacity of yeast fermentation potato glucose medium

The yeast fermentation medium solution is changed to potato dextrose (PDA) medium, which is an aqueous solution composed of the following components. The content of each component is respectively potato 200.0 and glucose 20.0 in g/1000mL aqueous solution, and the balance is water, pH The value is natural.

The preparation method is to weigh 200g of potatoes, wash, peel and cut into small pieces, add water and boil until rotten (boil for 20-30 minutes, it can be pierced by a glass rod), filter with four layers of gauze, add glucose, continue to heat and stir Mix well, cool down a bit, then add water to 1000 ml, divide into test tubes, stopper, bandage, sterilize at 112°C for 20 minutes, take out and cool.

Except for the culture medium and its production method, the culture period is 48 hours, and the sampling time interval is 4 hours, others such as culture temperature, shaking rate, concentration of inoculated viable bacteria, growth curve drawing, test tube liquid filling, inoculation and culture, antibiotic mother solution preparation , ethanol fermentation and primary screening of high ethanol-producing strains, primary screening result verification, etc. are all identical to Example 1.

Figure 4 shows the growth curve of Saccharomyces cerevisiae QD in PDA culture medium. It can be seen that the lag phase of strain growth is 0-4h, the logarithmic growth phase is 4-24h, and enters the stable phase after 24h; The growth curve of saccharomyces 2.1621 in PDA culture medium shows that the lag phase of strain growth is 0-4h, the logarithmic growth phase is 4-24h, and enters the stable phase after 24h; From the growth curve in the liquid, it can be seen that the lag phase of strain growth is 0-4h, the logarithmic growth phase is 4-24h, and enters the stable phase after 24h.

The following table 3 shows the bubble production of each strain

Note: According to the amount of gas produced in the fermentation tube, it is divided into 5 grades:

a "++++" indicates that the fermentation tube is filled with gas,

b "+++" means that the fermentation tube is filled with 3/4 gas,

c "++" means that the fermentation tube is filled with 1/2 gas,

d "+" indicates that the fermentation tube is filled with 1/4 gas,

e "-" indicates that there is no gas in the fermentation tube

Verification of preliminary screening results

In the PDA medium, the bubble production of each strain was basically similar to that in the YEPD medium, and Saccharomyces cerevisiae QD still produced more bubbles. The above-mentioned strains were analyzed for ethanol production to verify the correctness of the preliminary screening results. Get the following table 4 data

Note: The ethanol production value in the above table is measured after the fermentation stock solution is diluted 40 times

It can be seen from the data in Table 4 that in the PDA medium, the ethanol yield of Saccharomyces cerevisiae QD is still higher, which verifies the preliminary screening results listed in Table 3, indicating that the experimental scheme is correct.

Example 3: Preliminary judgment on ethanol-producing ability of yeast fermented bean sprouts juice glucose medium

The yeast fermentation medium solution is changed to bean sprouts juice glucose medium, which is an aqueous solution composed of the following components. The content of each component is calculated as g/1000mL aqueous solution: soybean sprouts 100.0, glucose 50.0, the balance is water, and the pH value nature.

Preparation method Weigh 100g of fresh soybean sprouts, put them in a beaker, add 1000 mL of water, boil on low heat for 30 minutes, filter with gauze, make up for the lost water, and then make 10% bean sprouts juice. Add 5g of glucose per 100mL of 10% bean sprout juice. Then subpackage, stopper, and bandage. Take out after high-pressure steam sterilization at 112°C for 20 minutes.

In addition to the difference in the culture medium and its production methods, others such as culture conditions, sampling time intervals, concentration of inoculated viable bacteria, growth curve drawing, test tube liquid distribution, inoculation and cultivation, antibiotic mother solution preparation, ethanol fermentation and preliminary screening of high ethanol-producing strains etc. are all identical with embodiment 2.

Accompanying drawing 7 is the growth curve of Saccharomyces cerevisiae QD in the bean sprouts juice glucose culture medium, it can be seen that the lag phase of strain growth is 0-4h, the logarithmic growth phase is 4-24h, and enters the stable phase after 24h; Accompanying drawing 8 is millet wine The growth curve of Schizosaccharomyces 2.1621 in PDA nutrient solution shows that the lag phase of strain growth is 0-4h, the logarithmic growth phase is 4-24h, and enters the stable phase after 24h; Accompanying drawing 9 is Kluyveromyces marx 191l From the growth curve in the PDA culture medium, it can be seen that the lag phase of strain growth is 0-4h, the logarithmic growth phase is 4-24h, and enters the stable phase after 24h.

The following table 5 is the bubble production situation of each bacterial strain

Note: According to the amount of gas produced in the fermentation tube, it is divided into 5 grades:

a "++++" indicates that the fermentation tube is filled with gas,

b "+++" means that the fermentation tube is filled with 3/4 gas,

c "++" means that the fermentation tube is filled with 1/2 gas,

d "+" indicates that the fermentation tube is filled with 1/4 gas,

e "-" indicates that there is no gas in the fermentation tube

Verification of preliminary screening results

In the bean sprouts juice glucose medium, the bubble production of each strain is basically similar to that in the YEPD medium, and Saccharomyces cerevisiae QD still produces more bubbles. The above-mentioned strains were analyzed for ethanol production to verify the validity of the preliminary screening results. Correctness, get the following table 6 data

Note: The ethanol production value in the above table is measured after the fermentation stock solution is diluted 40 times

From the data in Table 6, it can be seen that in the bean sprouts juice glucose medium, the ethanol yield of Saccharomyces cerevisiae QD is still higher. The results of quantitative analysis verified the preliminary screening results in Table 5, indicating that the experimental plan is correct.

Claims (3)

1. A method for rapid screening of high ethanol-producing yeast, characterized in that: the steps of the method are: Step 1, first draw the growth curve of the bacterial strain, and judge its lag phase, logarithmic growth phase, and stable phase accordingly; Step 2, preparing and distributing liquid culture medium in several test tubes, putting half of them upside down into a small fermentation tube filled with liquid medium, autoclaving the two test tube culture media, taking them out and cooling them; Step 3: Insert the strains into the test tube liquid culture medium without the fermentation tube, shake the culture until the end of logarithmic growth, and take it out for use; Step 4, during bacterial culture, prepare antibiotic mother solution, filter and sterilize; Step 5, aseptic operation Take out the fermentation tube in step 2 from the test tube liquid culture medium, put it upside down into the culture medium cultivated in the above step 3 to the end of logarithmic growth, ensure that there are no air bubbles in the tube, and add Appropriate amount of antibiotic solution prepared in the 4th step is transferred to static fermentation culture; Step 6. After culturing for one cycle, take out the test tube liquid in the above step 5, and observe the size of the air column in the fermentation tube. The larger the air column, the higher the ethanol production, so that the target strain can be quickly screened out. 2. The method according to claim 1, characterized in that the specific operations of the method are as follows: (1) Drawing of yeast growth curve Under the optimal culture conditions determined in previous experiments, including culture medium composition and content, culture temperature, pH, culture cycle, shaking speed, inoculate yeast in liquid medium and culture, and the culture time is regarded as horizontal. Coordinates, taking the absorbance value _A600 of the bacterium liquid as the ordinate, draw the growth curve of the absorbance value _A600 of the yeast culture solution changing with the culture time, and judge the lag period, logarithmic growth period, and stable period of bacterial strain growth accordingly; (2) Preparation and sterilization of test tube liquid medium Prepare 1000mL of glucose-containing medium YEPD or medium PDA that can be converted into glucose or bean sprout juice glucose medium or wort glucose medium, and divide it into 100 test tube liquids, each 9.5m ~ 9.8mL, take 50 of them Put a test tube liquid into it, put it upside down into a fermentation tube filled with 0.2mL~0.5mL of the above-mentioned liquid medium, carefully operate to avoid air bubbles in the small tube, record the test tube liquid medium with the fermentation tube as a, and the other 50 The liquid culture medium in test tubes without fermentation tubes is denoted as b, and at the same time, it is sterilized under 112°C and 0.5Mpa hot and humid steam for 20min, and it is taken out and cooled to room temperature; (3) Inoculation and cultivation Inoculate the yeast strain into the test tube liquid medium b without a fermentation tube with an inoculum amount of 0.5%, and culture it with shaking at 150 r/min at 30°C until the end of the logarithmic growth phase, take it out for use, and record it as c; (4) Preparation of antibiotic mother solution Prepare kanamycin solution mother liquor and chloramphenicol solution mother liquor respectively, concentration is 0.5g/100mL, two kinds of antibiotic mother liquors are filtered and sterilized under aseptic operation respectively; The preparation of antibiotic mother liquor is carried out in the bacteria liquid cultivation stage after inoculation; (5) Ethanol fermentation Under aseptic operation, take out the fermentation tube in a from the liquid in the test tube, quickly put it upside down and put it into the culture medium c cultivated to the end of the logarithmic growth phase, operate carefully so that no air bubbles enter the fermentation tube, and then rapidly transfer to the same tube Add kanamycin solution with a final concentration of 50 μg/mL and chloramphenicol solution with a final concentration of 25 μg/mL to the culture solution. Static culture to one culture cycle; A culture cycle includes a cell growth stage that does not produce ethanol and a cell fermentation glucose to produce ethanol and CO ₂ stage; (6) Primary screening of ethanol-producing strains Take out the above-mentioned culture solution c that has completed one culture cycle, observe the bubble generation and discharge of liquid in the fermentation tubule in the test tube liquid, and judge the amount of ethanol produced by the yeast according to the size of the air column in the fermentation tubule. Larger, which indirectly indicates that the ethanol production is higher, so that the target strain with high ethanol production can be screened out very quickly; the amount of ethanol production is determined by further quantitative detection. 3. The method according to claim 2, characterized in that the method further comprises: (7) Verification of preliminary screening results The high-yielding strains obtained from the above primary screening were subjected to ethanol-producing fermentation, and then samples were taken, diluted appropriately, and analyzed for ethanol production by using a biosensing analyzer or liquid chromatography to verify the accuracy of the primary screening results of this method.