Dynamic adaptive evolution method for improving high ethanol tolerance of saccharomyces cerevisiae and anaerobic chemostat culture device thereof

Abstract

The invention provides a dynamic adaptive evolution method for improving high ethanol tolerance of saccharomyces cerevisiae and an anaerobic chemostat culture device thereof, and belongs to the technical field of microorganisms. According to the method, ethanol is added into a culture medium in a gradient manner, and the saccharomyces cerevisiae is subjected to adaptive evolution in an anaerobic chemostat system. The evolved strain significantly improves the tolerance to ethanol. When the evolutionary strain is applied to production of ethanol under different fermentation conditions in a high-glucose environment, the substrate metabolism capability, unit respiration capability and ethanol product synthesis capability of unit thalli are all improved, and ethanol can be efficiently synthesized by adopting the adaptive evolutionary saccharomyces cerevisiae strain for fermentation production of ethanol.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a dynamic adaptive evolution method for improving the high ethanol tolerance of brewer's yeast and an anaerobic constant culture device thereof. Technical Background

[0002] Fuel ethanol is currently the most widely used renewable bio-liquid fuel in the world. It can be used as an environmentally friendly oxygenator and octane enhancer for gasoline.

[0003] With the continuous reduction of global petrochemical resources and the increasing awareness of environmental protection, the development of fuel ethanol as a substitute for traditional petrochemical energy has attracted more and more attention. In the process of industrial alcohol fermentation, alcohol inhibition refers to the inhibitory effect of ethanol on the growth and alcohol production ability of yeast cells when the ethanol concentration reaches a certain level. This inhibitory effect may affect the vitality, metabolic activity and fermentation effect of yeast cells, so it is urgent to develop a high ethanol tolerance strain to improve fermentation efficiency. Summary of the invention

[0004] The present application provides a dynamic adaptive evolution method and an anaerobic constant culture device for improving the high ethanol tolerance of brewer's yeast, and the method can be used to adaptively evolve brewer's yeast by adding ethanol in a gradient manner to the culture medium. The evolved strain significantly improves the tolerance to ethanol. The evolved strain is applied to produce ethanol under different fermentation conditions in a high-sugar environment, and the substrate metabolism capacity per bacterial body, the unit respiration capacity and the ethanol product synthesis capacity are all improved. The use of the adaptively evolved brewer's yeast strain obtained in the present application to produce ethanol by fermentation can efficiently synthesize ethanol, which is of great significance for industrial production.

[0005] The present application provides a dynamic adaptive evolution method for improving the high ethanol tolerance of Saccharomyces cerevisiae, which comprises:

[0006] (1) inoculating the cultured brewer's yeast solution into a batch culture system for batch culture;

[0007] (2) At the end of the batch culture, the culture system is switched to a chemostat culture system for chemostat culture: ethanol containing an initial concentration is added to the chemostat culture system at a constant rate to maintain a constant volume of the culture system;

[0008] (3) Obtaining the fermentation tail gas results during the adaptive evolution process, using the fermentation tail gas component detection, combined with the changes in the process biological parameters to determine and adjust the ethanol concentration, and dynamically adjusting the adaptive concentration through the dilution rate and ethanol concentration changes, and gradually increasing it to the target concentration;

[0009] (4) A target strain that maintains a constant specific growth rate in a target concentration of ethanol is obtained, and a cerevisiae yeast strain with high ethanol tolerance is obtained after purification and screening.

[0010] Optionally, in some embodiments of the present application, the initial concentration is 25 g / L-35 g / L; the target concentration is 65 g / L-75 g / L;

[0011] Optionally, the ethanol concentration is adjusted by detecting the components of the fermentation tail gas in combination with the changes in the biological parameters of the process, the carbon dioxide content of the tail gas is obtained by detecting the components of the tail gas, the CER value is calculated, the CER threshold is set to 10-25 mmol / l / h, and the biomass threshold is set to 1.5-2.5 g / kg, and the CER value and the biomass value are compared with the CER threshold and the biomass threshold, respectively. After 3-5 elution volumes, when the CER value and the biomass value are respectively maintained within the range of the CER threshold and the biomass threshold and remain stable, adaptive evolution of higher ethanol concentration is performed;

[0012] Optionally, when the CER value and the biomass value are not within the CER threshold value and the biomass threshold value range and there is a continuous decline phenomenon, the ethanol concentration or the dilution rate is dynamically adjusted to perform adaptive evolution.

[0013] The present application provides an anaerobic chemostat culture device, which is used for the above-mentioned adaptive evolution method for improving the resistance of Saccharomyces cerevisiae to ethanol to perform chemostat culture evolution on Saccharomyces cerevisiae, and comprises:

[0014] The reactor provides a sterile anaerobic environment for the culture system by introducing high-purity nitrogen, and the culture is carried out by configuring a stirring component;

[0015] A feeding part, adding a chemostatic medium containing different concentrations of ethanol into the reactor at a determined rate;

[0016] A discharge part, used to discharge the fermentation liquid exceeding a set volume in the reactor;

[0017] The gas detection unit obtains the fermentation tail gas of the reactor and detects the carbon dioxide content of the fermentation tail gas for judging the current ethanol adaptive evolution process.

[0018] Optionally, in some embodiments of the present application, the feeding unit includes:

[0019] A first storage container is used to store a chemostat culture medium;

[0020] A first pipeline, one end of which is connected to the culture solution storage container, and the other end of which is connected to the reactor, wherein the first pipeline is provided with a first peristaltic pump for pumping the chemostatic culture medium into the reactor;

[0021] A second storage container, for ethanol solutions of different concentrations;

[0022] A second pipeline has one end connected to the culture solution storage container and the other end connected to the reactor. A second peristaltic pump for pumping the chemostatic culture medium into the reactor is provided on the second pipeline.

[0023] Optionally, in some embodiments of the present application, the discharge portion includes:

[0024] A third storage container, for receiving the discharged fermentation liquid;

[0025] A third pipeline has one end connected to the culture solution storage container and the other end connected to the reactor. The third pipeline is provided with a third peristaltic pump for pumping the chemostatic culture medium into the reactor.

[0026] Optionally, in some embodiments of the present application, the gas detection unit includes:

[0027] A mass spectrometer for detecting the components of fermentation tail gas; wherein the fermentation tail gas in the reactor is sent to the mass spectrometer for detection.

[0028] The present application provides the application of a high ethanol-tolerant Saccharomyces cerevisiae strain in different fermentation modes under high-sugar culture medium conditions.

[0029] The present application has at least one or more of the following beneficial effects:

[0030] In this application, the brewer's yeast that has not been subjected to mutagenesis treatment is subjected to an adaptive evolution experiment by adding exogenous ethanol to facilitate the subsequent exploration of resistance genes and multi-omics comprehensive analysis.

[0031] In the present application, the adaptive evolution of constant culture under suitable conditions for the bacteria can make the bacteria evolve in a relatively stable state, reduce the uncontrollable adverse factors that may occur under other culture conditions, and enable the bacteria to evolve stably under suitable cell density and ethanol evolution pressure, thereby improving the evolution efficiency.

[0032] In this application, an anaerobic environment is created by nitrogen to simulate the process environment of industrial ethanol production by brewer's yeast. The strain obtained in this application is more adaptable to actual industrial production conditions, improves the strain's ethanol resistance under actual production conditions, and improves ethanol production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Changes in parameter settings for the adaptive evolution process (a) shows changes in exogenous ethanol concentration, and b shows changes in continuous culture dilution rate);

[0034] Figure 2The changes of macro parameters in the adaptive evolution process (a represents cell dry weight, b represents carbon dioxide release rate CER, c represents glycerol concentration, d represents glucose concentration, and e represents ethanol concentration);

[0035] Figure 3 The biomass of different strains under different ethanol concentrations (Figure a shows the growth of strains under 0% ethanol conditions, Figure b shows the growth of strains under 7.5% ethanol conditions, and Figure c shows the growth of strains under 12% ethanol conditions);

[0036] Figure 4 The results of high-sugar batch culture of different strains (a) show cell dry weight, b show CER, c show glucose concentration, d show glycerol concentration, and e show ethanol concentration);

[0037] Figure 5 The results of high-sugar fed-batch culture of different strains (a) show cell dry weight, b show CER, c show glucose concentration, d show glycerol concentration, and e show ethanol concentration);

[0038] Figure 6 Schematic diagram of the structure of the constant temperature culture device. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present application. In addition, it should be understood that the specific implementation methods described herein are only used to illustrate and explain the present application and are not used to limit the present application. In the present application, unless otherwise stated, the directional words used, such as "up", "down", "left", and "right", generally refer to the up, down, left, and right of the device in actual use or working state, specifically the drawing direction in the accompanying drawings.

[0040] The present application provides a dynamic adaptive evolution method for improving the high ethanol tolerance of brewer's yeast and an anaerobic chemostat culture device thereof, which are described in detail below. It should be noted that the description order of the following embodiments is not intended to limit the preferred order of the embodiments of the present application. In the following embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, please refer to the relevant description of other embodiments.

[0041] The present application example provides a dynamic adaptive evolution method for improving the high ethanol tolerance of Saccharomyces cerevisiae, and the steps are as follows:

[0042] S1, inoculating the cultured brewer's yeast solution into a batch culture system at a 10% inoculation rate for batch culture;

[0043] S2, at the end of the batch culture, switching the culture system to a chemostat culture system for chemostat culture: adding ethanol containing an initial concentration to the chemostat culture system at a constant rate to keep the culture system at a constant volume;

[0044] Cultivate evolution, specifically: add the chemostat medium to the culture system at a determined rate, and control the specific growth rate at 0.055h -1 -0.1h -1 Excess fermentation liquid was removed to maintain a constant volume of the culture system.

[0045] S3, obtain the fermentation tail gas results during the adaptive evolution process, use the fermentation tail gas component detection, combine the process biological parameter changes to determine the adjustment of ethanol concentration, and dynamically adjust the adaptive concentration through the dilution rate and ethanol concentration changes, and gradually increase it to the target concentration. The initial concentration is 25-35g / L; the optimal initial concentration is 30g / L, which is a suitable evolutionary pressure starting point obtained from the preliminary experiment of the starting strain.

[0046] S4, obtaining a target strain that maintains a constant specific growth rate in a target concentration of ethanol, and obtaining a high ethanol-tolerant Saccharomyces cerevisiae strain after purification and screening.

[0047] The target concentration is set to 20g / L-80g / L, and the most preferred target concentration is 65g / L-70g / L. When the concentration is higher than 70g / L, it takes longer to evolve. The most preferred ethanol concentration range is 30g / L-70g / L.

[0048] In some embodiments, the components of the batch culture medium include: 1g / L-4g / L KH2PO4, 3.5g / L-6.5g / L (NH4)2SO4, 0.4g / L-0.6g / L MgSO4·7H2O, 20.0g / L-30.0g / L glucose, 0.5mL / L-1.5mL / L metal ion stock solution, 0.5mL / L-1.5mL / L vitamin stock solution, 0.2g / L-0.5g / L Tween 80, 50μg / L-150μg / L ergosterol and 0.5mL / L-1.05mL / L defoaming agent. In one specific example, the components of the batch culture medium include: 3 g / L KH2PO4, 5 g / L (NH4)2SO4, 0.5 g / L MgSO4·7H2O, 25 g / L glucose, 1 mL / L metal ion stock solution, 1 mL / L vitamin stock solution, 0.4 g / L Tween 80, 100 μg / L ergosterol and 1 mL / L defoaming agent.

[0049] In some embodiments, the chemostat culture components include: 2g / L-5g / L KH2PO4, 3g / L-6g / L (NH4)2SO4, 0.3g / L-0.7g / L MgSO4·7H2O, 20.0g / L-30.0g / L glucose, 0.4mL / L-1.4mL / L metal ion stock solution, 0.4mL / L-1.4mL / L vitamin stock solution, 0.1g / L-0.6g / L Tween 80, 50μg / L-150μg / L ergosterol, 0.5mL / L-1.05mL / L defoaming agent and 20g / L-80g / L ethanol. In one specific example, the components of the batch culture medium include: 3g / L KH2PO4, 5g / L (NH4)2SO4, 0.5g / L MgSO4·7H2O, 25g / L glucose, 1mL / L metal ion stock solution, 1mL / L vitamin stock solution, 0.4g / L Tween 80, 100μg / L ergosterol, 1mL / L defoaming agent and 30-70g / L ethanol.

[0050] The metal ion components include: 19.0g / L EDTA, 4.5g / L ZnSO4·7H2O, 4.5g / L CaCl2, 3.0g / L FeSO4·7H2O, 1.0g / L H3BO3, 1.0g / L MnCl2·4H2O, 0.4g / L Na2MoO4·2H2O, 0.3g / L CoCl2·6H2O, 0.3g / L CuSO4·5H2O and 0.1g / L KI; the pH is adjusted to 4.0 with 2mol / L HCl. The vitamin mother solution formula is 50mg / L biotin, 1000mg / L pantothenic acid hemicalcium salt, 1000mg / L thiamine hydrochloride, 1000mg / L pyridoxine hydrochloride, 1000mg / L nicotinic acid, 200mg / L p-aminobenzoic acid and 25000mg / L inositol.

[0051] In some embodiments of the present application, before inoculating the brewer's yeast in batch culture, the process further includes: culturing to obtain the brewer's yeast, the culturing process including: inoculating the activated strain into a seed culture medium, culturing at a speed of 210 rpm-230 rpm and a temperature of 28° C.-32° C. for 10-14 hours; most preferably, the speed is 220 rpm, the temperature is 30° C., and the culturing time is 12 hours;

[0052] The components of the seed culture medium include: 10g / L-15g / L KH2PO4, 5g / L-8g / L (NH4)2SO4, 0.3g / L-0.7g / L MgSO4·7H2O, 5g / L-15g / L glucose, 1.5mL / L-2.5mL / L metal ion mother solution and 0.5mL / L-1.5mL / L vitamin mother solution. In a specific example, the components of the seed culture medium include: 14.4g / L KH2PO4, 7.5g / L (NH4)2SO4, 0.5g / L MgSO4·7H2O, 10g / L glucose, 2mL / L metal ion mother solution and 1mL / L-1.4mL / L vitamin mother solution.

[0053] The present application provides an anaerobic constant temperature culture device, which is used for constant temperature culture of Saccharomyces cerevisiae by the above-mentioned adaptive evolution method for improving the resistance of Saccharomyces cerevisiae to ethanol, and comprises: a reactor, a feeding part, an outflow part and a gas detection part.

[0054] The reactor provides a sterile anaerobic environment for the culture system. A stirring component for stirring the culture system is provided in the reactor. The stirring component can be a magnetically driven stirring system. The reactor is a miniature bioreactor. The volume of the culture system is controlled at 3000mL, and a 5000mL reactor is selected. The reactor can be, but is not limited to, a 5000mL parallel reactor, which can be selected from Shanghai Guoqiang Biochemical Engineering Equipment Co., Ltd.

[0055] The feeding section is used to add chemostatic culture medium and ethanol containing different concentrations (i.e., feeding solution) into the reactor at a determined rate.

[0056] The gas detection unit obtains the fermentation exhaust gas in the reactor and performs real-time detection and analysis on its components to obtain the CER value used to determine the completion of the current ethanol concentration evolution. The trend change of the CER value determines whether it is necessary to adjust the ethanol concentration sent by the feed unit, which serves as the basis for whether it is necessary to adjust the ethanol concentration in the feed unit.

[0057] The above device is used for chemostat culture:

[0058] The batch culture medium is placed in the reactor, and the saccharomyces cerevisiae liquid is inoculated into the batch culture medium in the reactor, and the inoculation amount can be 10%. The temperature is maintained at 30°C by the heating base and the condensation sleeve at the bottom of the reactor. The batch culture and the constant temperature culture in the subsequent evolution process are carried out in a 5000ml reactor with a culture volume of 3000ml. The preferred conditions are: the ventilation rate of sterile high-purity nitrogen gas in the reactor is 0.5vvm, and the stirring speed is 150rpm.

[0059] The present application provides a high ethanol tolerance Saccharomyces cerevisiae strain obtained after adaptive evolution, and provides an application of the high ethanol tolerance Saccharomyces cerevisiae strain, which includes:

[0060] (1) The cultured strains were inoculated into YPD medium supplemented with 0%, 7.5%, and 12% ethanol for batch fermentation, and the OD600 was measured to explore the tolerance of the screened strains to ethanol.

[0061] (2) The cultured strains were inoculated into a medium containing 285 g / L of glucose for high-sugar fermentation, simulating the VHG fermentation form in the factory, and exploring the fermentation metabolism of the strains before and after evolution in the high-sugar environment of the factory. The different culture conditions were batch fermentation and fed-batch fermentation. The initial sugar in fed-batch fermentation was set to 100 g / L. When the yeast consumed the sugar to a low sugar concentration (less than 30 g / L), the remaining glucose was added twice to control the residual sugar in the fermentation process to between 20 and 100 g / L, reducing the severe substrate inhibition caused by high-sugar batch fermentation, and analyzing the metabolism of the strains under different cultures.

[0062] In order to enable the above implementation details and operations of the present application to be clearly understood by those skilled in the art, as well as to demonstrate the significant improvement in the performance of the adaptive evolution method of high-ethanol-tolerant Saccharomyces cerevisiae based on chemostat culture in the embodiments of the present application, the above technical scheme is illustrated by multiple embodiments below.

[0063] Example 1

[0064] The specific steps of laboratory evolution of high ethanol tolerance Saccharomyces cerevisiae are as follows:

[0065] (1) Dilute the strain in the glycerol tube to 10 6 Then, 40 μL was spread on a YPD plate for activation, and the plate was inverted in a 30°C incubator for 48 h.

[0066] (2) In a clean bench, inoculate the vitamin into a seed culture shake flask, take 5 mL and inoculate it into a glass test tube, pick a single colony from the activated YPD plate and inoculate it into the test tube, culture it at 30°C, 220 rpm for 12 h until the logarithmic growth phase, then inoculate the cultured test tube bacteria into a 500 mL shake flask with a liquid volume of 100 mL, and culture it at 30°C, 220 rpm for 12 h until the logarithmic growth phase.

[0067] (3) Flame inoculation: The bacterial liquid and glucose solution were aseptically inoculated into a 5L fermentation tank, with an inoculation volume of 10%, a culture system of 3L, a temperature of 30°C, a rotation speed of 150rpm, and a 99.99% high-purity nitrogen gas, with a ventilation volume of 1.5L / min, a tank pressure of 0.05Mpa, and a pH of 5±0.1 controlled by 4M NaOH. The pH was measured online by a pH electrode, and pH calibration was performed using standard solutions of pH 4.04 and 6.86. The exhaust gas components were analyzed by process exhaust mass spectrometry, the proportion of carbon dioxide in the exhaust gas was analyzed online, and the carbon dioxide generation rate (CER) was calculated by the difference in the proportions in the intake and exhaust gas. When the CER decreased and the pH increased, it was determined that the substrate carbon source was exhausted, the batch fermentation was terminated, and feeding and waste discharge were started for continuous culture.

[0068] (4) The parameters of constant culture and batch culture are consistent. For constant culture, fresh culture medium should be added and discharged through the waste pipe on the liquid surface by peristaltic pump to control the volume in the fermenter to 3L. A magnetic stirrer is used to fully mix the culture medium in the feed bucket to ensure the consistency of the concentration of the culture medium in the bucket. The dilution rate D is controlled at 0.05-0.1h by adjusting the pump speed. -1 ,When parameters such as CER, bacterial concentration and residual sugar remain stable, it is considered that the constant culture has reached metabolic steady state.

[0069] (5) Obtain the fermentation tail gas results during the adaptive evolution process, use the fermentation tail gas composition detection, combine the process biological parameter changes to determine the adjustment of ethanol concentration, and dynamically adjust the adaptive concentration through the dilution rate and ethanol concentration changes, and gradually increase it to the target concentration of 70 g / L (such as Figure 1 , Figure 2 shown).

[0070] Example 2

[0071] Comparative experiment on strain growth before and after evolution under different ethanol conditions

[0072] In this example, the specific operations are as follows:

[0073] The strains before and after adaptation were cultured in YPD medium supplemented with 0%, 7.5%, and 12% for 12 h, and the OD was measured every 2 h (e.g. Figure 3As shown in Figure 2, the growth trends of the two strains are consistent. At 6h, the adapting bacteria are slightly lower than the starting bacteria, and this trend is maintained until 12h. The final biomass is 10.1 and 9.5 respectively, and the biomass of the adapting bacteria is about 7% lower than that of the starting bacteria. The reason for this situation may be that the growth of the bacterium is inhibited during the adaptive evolution of ethanol. During the adaptive evolution, the strain may adjust the metabolic pathway and physiological mechanism to adapt to the environment of high ethanol concentration, but these adjustments may have a negative impact on the growth rate or biomass accumulation. The final adaptive evolution strain may show a lower growth ability under the ethanol environment. The growth of the b figure with the addition of 7.5% ethanol can be clearly seen that the adapting strain has better growth survival ability than the starting bacteria under the condition of such inhibition, and the final biomass is about 25% higher. The c figure shows the growth of the c figure with the addition of 12% ethanol. In this experiment, 12% ethanol was added at 6h. Under the condition of lethal ethanol concentration, the starting bacteria showed stagnation when placed in high ethanol concentration, while the adapting bacteria can tolerate this concentration well, and the growth rate slows down but can continue to grow, and the final biomass is 20% higher than the starting bacteria. After adaptive evolution experiments, the yeast strain increased its tolerance to ethanol and showed better growth characteristics at inhibitory and lethal concentrations, achieving the goal of ethanol tolerance, proving the feasibility of this approach.

[0074] Example 3

[0075] Fermentation performance of adaptively evolved strains in different fermentation forms

[0076] In this example, the specific operations are as follows:

[0077] The starting bacteria were used as the control group, and the adaptively evolved bacteria were cultured in two fermentation modes: high-sugar batch fermentation and high-sugar fed-batch fermentation. The fermentation characteristics of the two in different fermentation modes were compared and analyzed to study the physiological metabolism (such as Figure 4 , Figure 5 shown).

[0078] In batch fermentation mode, the strains all grew rapidly in the first 12 hours. After 12 hours, the growth of the starting bacteria was still increasing slowly, while the evolved bacteria entered the stable period and maintained a stable biomass. The biomass of the starting bacteria and the adapted bacteria were 9.9g / kg and 9.0g / kg, respectively, with a difference of 10%. This may be due to the adaptation process of ethanol, the process metabolism changed, and there was no need to produce too much biomass to maintain the fermentation capacity. From the CER graph, it can be seen that the two strains grew rapidly in the first 12 hours and released a large amount of carbon dioxide, and then began to decline. The time for the adapted bacteria to reach the highest point was 2 hours faster, and there was a difference in metabolism between the two. The starting bacteria and the adapted bacteria had the same trend in glucose consumption, glycerol synthesis, and ethanol production. The final glycerol concentrations of the starting bacteria and the adapted bacteria were 9.6g / L and 9.9g / L, respectively, with a difference of 3%. The difference in glycerol synthesis may be due to the environmental pressure during the adaptation process of ethanol, which forced the bacteria to synthesize more glycerol to maintain cell vitality. This trait gradually became a stable genetic trait in the long-term evolution process. The total fermentation time of the adapted bacteria was 50 h, which was 6% shorter than the 53-hour fermentation time of the starting bacteria. This showed that the fermentation rate of the adapted bacteria was accelerated and the fermentation capacity per unit cell was improved to a certain extent.

[0079] In the fed-batch fermentation mode, the bacteria grow rapidly under the appropriate sugar concentration. The starting bacteria reach the highest dry weight of 12.1g / kg at 16h, while the adaptive bacteria only have a dry weight of 10.5g / kg. The difference between the two is 15%, which is a significant difference. The respiratory metabolic capacity of the starting bacteria reaches the highest point after 8h and then begins to decline, while the adaptive bacteria reach this state 1h earlier than the starting bacteria. The difference in bacterial quantity is also reflected in the absolute numerical difference of CER. Since the bacterial quantity of the adaptive bacteria is less than that of the starting bacteria, the highest point of CER is also lower than that of the starting bacteria. In this culture mode, there is no significant difference in the glucose consumption rate of the bacteria, and the synthesis rates of glycerol and ethanol, and they remain at the same level.

[0080] Table 1 Specific growth rates of adapted bacteria in the logarithmic growth period (8 h) under different culture conditions

[0081]

[0082] Two kinds of fermentation modes, what mainly changes is the metabolic mode of substrate glucose, therefore analyze two kinds of bacteria and carry out intracellular metabolic rate analysis in the growth period (8h) that batch fermentation and fed batch fermentation have significant differences.Under batch culture and fed batch culture conditions, the glucose of adapting bacteria is compared with the starting bacteria and improves by 10.8%, 3.3% respectively than the consumption rate, the specific generation rate of glycerol is reduced by 6.7%, improves by 10.4%, the specific generation rate of ethanol is improved by 3.3%, 14.2%, and the specific release rate of carbon dioxide is improved by 12.9%, 2.6%.It can be concluded that adapting bacteria has improved the substrate metabolism ability of unit thalline compared with adapting bacteria, has stronger unit respiration ability and ethanol product synthesis ability, and can be used as subsequent fermentation use.(as table 1).As can be seen from the above embodiments, the application carries out high ethanol tolerance strain breeding by laboratory adaptive evolution, and obtains high ethanol tolerance strain. The purpose of the study was to explore the effect of increased ethanol concentration on the macro-metabolic expression of Saccharomyces cerevisiae and changes in cell morphology, study and compare the growth of the bacteria in different ethanol environments before and after the experiment, and the ethanol fermentation performance under different fermentation forms, investigate the physiological metabolism of the adaptable bacteria, and study the macro-metabolic mechanism of ethanol tolerance of Saccharomyces cerevisiae. The obtained high ethanol-tolerant strain has better unit cell substrate conversion capacity and ethanol synthesis capacity than the starting bacteria, which is of great significance for industrial ethanol fermentation.

[0083] The above is a detailed introduction to the present application. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. At the same time, for general technical personnel in this field, according to the idea of ​​the present application, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A dynamic adaptive evolution method for improving the high ethanol tolerance of Saccharomyces cerevisiae, characterized in that: It includes: (1) inoculating the cultured brewer's yeast solution into a batch culture system for batch culture; (2) At the end of the batch culture, the culture system is switched to a chemostat culture system for chemostat culture: ethanol containing an initial concentration is added to the chemostat culture system at a constant rate to maintain a constant volume of the culture system; (3) Obtaining the fermentation tail gas results during the adaptive evolution process, using the fermentation tail gas component detection, combined with the changes in the process biological parameters to determine and adjust the ethanol concentration, and dynamically adjusting the adaptive concentration through the dilution rate and ethanol concentration changes, and gradually increasing it to the target concentration; (4) A target strain that maintains a constant specific growth rate in a target concentration of ethanol is obtained, and a cerevisiae yeast strain with high ethanol tolerance is obtained after purification and screening.

2. A dynamic adaptive evolution method for improving high ethanol tolerance of Saccharomyces cerevisiae according to claim 1, characterized in that: The initial concentration is 25g / L-35g / L; the target concentration is 65g / L-75g / L.

3. The method of utilizing fermentation tail gas component detection and combining process biological parameter changes to determine and adjust ethanol concentration according to claim 1, characterized in that: The carbon dioxide content of the tail gas is obtained by detecting the tail gas components, and the CER value is calculated. The CER threshold is set to 10-25 mmol / l / h, and the biomass threshold is set to 1.5-2.5 g / kg. The CER value and the biomass value are compared with the CER threshold and the biomass threshold, respectively. After 3-5 elution volumes, when the CER value and the biomass value are respectively maintained within the CER threshold and the biomass threshold range and remain stable, a higher concentration of ethanol adaptive evolution is performed; When the CER value and the biomass value are not within the CER threshold value and the biomass threshold value range and there is a continuous decline phenomenon, the ethanol concentration or the dilution rate is dynamically adjusted to perform adaptive evolution.

4. An anaerobic constant temperature culture device, characterized in that: The adaptive evolution method for improving the ethanol resistance of Saccharomyces cerevisiae according to any one of claims 1 to 5 comprises: subjecting the Saccharomyces cerevisiae to anaerobic chemostat culture evolution, which comprises: The reactor provides a sterile anaerobic environment for the culture system by introducing high-purity nitrogen, and the culture is carried out by configuring a stirring component; A feeding part, adding a chemostatic medium containing different concentrations of ethanol into the reactor at a determined rate; A discharge part, used to discharge the fermentation liquid exceeding a set volume in the reactor; The gas detection unit obtains the fermentation tail gas of the reactor and detects the carbon dioxide content of the fermentation tail gas for judging the current ethanol adaptive evolution process.

5. The anaerobic chemostat culture device according to claim 4, characterized in that: The feeding unit comprises: A first storage container is used to store a chemostat culture medium; A first pipeline, one end of which is connected to the culture solution storage container, and the other end of which is connected to the reactor, wherein the first pipeline is provided with a first peristaltic pump for pumping the chemostatic culture medium into the reactor; A second storage container, for ethanol solutions of different concentrations; A second pipeline has one end connected to the culture solution storage container and the other end connected to the reactor. A second peristaltic pump for pumping the chemostatic culture medium into the reactor is provided on the second pipeline.

6. The anaerobic chemostat culture device according to claim 4, characterized in that: The discharge unit comprises: A third storage container, for receiving the discharged fermentation liquid; A third pipeline has one end connected to the culture solution storage container and the other end connected to the reactor. The third pipeline is provided with a third peristaltic pump for pumping the chemostatic culture medium into the reactor.

7. The anaerobic chemostat culture device according to claim 4, characterized in that: The gas detection unit comprises: A mass spectrometer for detecting the components of fermentation tail gas; wherein the fermentation tail gas in the reactor is sent to the mass spectrometer for detection.

8. Use of the high ethanol tolerance Saccharomyces cerevisiae strain obtained as claimed in claim 1 in different fermentation modes under high sugar medium conditions.

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