A method for regulating bacillus subtilis growth for spore production using a redox electrode

Abstract

The application discloses a method for regulating growth of bacillus subtilis by using a redox electrode to produce spores, and belongs to the technical field of fermentation engineering. The method for regulating growth of bacillus subtilis by using a redox electrode to produce spores comprises the following steps: in the process of fermentation production, a redox electrode is added, and the redox potential of the bacillus subtilis fermentation liquor is controlled to be in the range of 50-350 mV. The method can make the bacillus subtilis grow and produce spores at a faster rate, significantly improve the spore yield and spore rate, and effectively improve the carbon source substrate conversion rate and air utilization rate.

Description

Technical Field

[0001] This application relates to the field of fermentation engineering technology, specifically to a method for spore production by regulating the growth of Bacillus subtilis using a redox electrode. Background Technology

[0002] Bacillus subtilis, as one of the biocontrol bacteria for plant diseases, has a strong disease prevention effect. For example, it can be used to control diseases such as powdery mildew, downy mildew, blight, and gray mold in various crops. Bacillus subtilis can also be used as a feed microbial strain to prepare microbial additives to improve animal intestinal function, promote animal growth, and prevent diseases. In aquaculture, it can effectively reduce the concentration of amino nitrogen, nitrite nitrogen, and sulfides in water, thereby effectively improving water quality. It can also provide nutrients for phytoplankton, mainly single-celled algae, and the various enzymes and antibiotics secreted by them can inhibit the growth of other bacteria, thereby reducing or even eliminating pathogens in aquatic animals. Bacillus subtilis can also be used as a novel microbial biological pesticide, with good effects on the control of diseases in crops such as rice, wheat, peanuts, tomatoes, peppers, soybeans, and corn, especially effective against wheat powdery mildew, Fusarium head blight, and sheath blight.

[0003] It is evident that Bacillus subtilis has a wide range of applications and a large demand. Since Bacillus subtilis spores are dormant bacterial bodies, they have better transport and preservation characteristics compared to Bacillus subtilis growing bodies. Therefore, there is an urgent need for a method that can efficiently grow Bacillus subtilis and produce spores to meet the growing market demand. Existing technologies mostly involve artificially controlling the redox potential during fermentation to shift the redox balance in a direction favorable to the accumulation of the target product. For example, in the field of biochemical engineering, by selecting suitable cells and controlling the redox potential for fermentation production, the yields of metabolites such as succinic acid, propylene glycol, citric acid, hydrogen, clavulanic acid, hexanal, xylitol, and ethanol can be increased. However, there is very little research on controlling the redox potential to promote cell growth and spore production in Bacillus subtilis. Moreover, compared to existing methods that only require controlling the redox potential to increase the yield of a single metabolite, the production process of Bacillus subtilis is more complex. It requires controlling cell growth to increase the initial number of cells and regulating the spore transformation process to increase the yield of the final product, spores. Therefore, a method for producing Bacillus subtilis spores by controlling the redox potential is essential.

[0004] It should be noted that the content involved in the background art described above in this application does not necessarily constitute prior art. Summary of the Invention

[0005] To address the aforementioned issues, this application provides a method for spore production by regulating the growth of Bacillus subtilis using a redox electrode. The method provided in this application can significantly increase spore yield and spore rate by adjusting the fermentation process of Bacillus subtilis in spore production, thereby improving the conversion rate of carbon source matrix and air utilization rate, increasing output, saving costs, improving resource utilization, and exhibiting good process stability.

[0006] According to one aspect of this application, a method for spore production of Bacillus subtilis by regulating its growth using a redox electrode is provided. During fermentation, the redox potential of the Bacillus subtilis fermentation broth is controlled within the range of 50–350 mV. The method provided in this application, by controlling the redox potential of the Bacillus subtilis fermentation broth within a certain range during fermentation, can increase spore yield compared to conventional methods that do not control the redox potential. This allows for intervention and influence on the growth and spore production process of Bacillus subtilis through potential control.

[0007] Optionally, the fermentation process is divided into a first fermentation stage and a second fermentation stage. In the first fermentation stage, the oxidation-reduction potential of the fermentation broth is controlled within the range of 150 to 350 mV. In the second fermentation stage, the oxidation-reduction potential of the fermentation broth is controlled within the range of 50 to 150 mV. The ratio of the fermentation time of the first fermentation stage to the fermentation time of the second fermentation stage is 1:0.8 to 1.2.

[0008] The redox potential in the fermentation broth affects the growth of Bacillus subtilis, while the growth of Bacillus subtilis also affects the redox potential in the fermentation broth. By controlling the redox potential of the fermentation broth in the first fermentation stage to a range of 150–350 mV, the optimal growth rate of Bacillus subtilis can be ensured, thereby obtaining the maximum cell mass. In the second fermentation stage, controlling the redox potential of the fermentation broth in the range of 50–150 mV can reduce the rate at which Bacillus subtilis utilizes reducing sugars in the fermentation broth, thereby reducing the concentration of GTP / GMP in the fermentation broth and promoting the generation and maturation of spores in the fermentation broth.

[0009] Optionally, the redox potential of the fermentation broth is controlled within the range of 245–255 mV in the first fermentation stage, and within the range of 95–105 mV in the second fermentation stage. By further narrowing the fluctuation range of the redox potential, the redox potential can be kept stable during fermentation, thereby stabilizing the growth and spore production process of Bacillus subtilis, which is more conducive to improving the spore yield of Bacillus subtilis. Furthermore, setting the potential range of 245–255 mV in the first fermentation stage can also effectively reduce the cost of maintaining a high potential.

[0010] Optionally, in the fermentation production process, the total fermentation time of the first fermentation stage and the second fermentation stage is 30 to 38 hours.

[0011] By controlling the fermentation time within the aforementioned range, high production capacity and high conversion rate can be achieved while improving air utilization. If the first fermentation stage is too short, the bacterial count in the fermentation broth will be low, failing to reach optimal production capacity. Conversely, if the first fermentation stage is too long, the bacterial count will be high, and some cells may undergo autolysis, resulting in a low nutrient concentration in the culture medium, which cannot meet the nutrient requirements of the second fermentation stage. If the second fermentation stage is too short, the spore rate in the fermentation broth will be low, leading to low carbon source conversion. Conversely, if the second stage is too long, it will result in resource waste and reduced energy and air utilization.

[0012] Optionally, the temperature of the fermentation broth is 36–39°C. Within the range of 36–39°C, Bacillus subtilis can achieve the maximum growth rate, shorten the fermentation cycle, and achieve a high spore formation rate and a large spore quantity. Below this temperature, the growth rate of Bacillus subtilis is low, the bacterial count is small, the spore formation rate is slow, the fermentation cycle is long, and the production cost is high. Above this temperature range, the growth rate of Bacillus subtilis is too fast, which can easily cause autolysis of the bacteria, premature spore formation, and reduced overall spore yield. Moreover, maintaining a higher temperature increases heat costs, thereby increasing the overall production cost.

[0013] Optionally, the method for controlling the redox potential of the fermentation broth is as follows:

[0014] (1) Measure the redox potential of the fermentation broth and set the initial stirring speed and initial aeration rate;

[0015] (2) When the redox potential is lower than the lower limit of the redox potential range, increase the stirring speed of the fermentation broth and the air flow rate; when the redox potential is higher than the upper limit of the redox potential range, decrease the stirring speed of the fermentation broth and the air flow rate.

[0016] By controlling the stirring speed and air flow rate of the fermentation broth, the direction of change in redox potential can be controlled. Higher stirring speeds and air flow rates tend to increase the redox potential, while lower stirring speeds and air flow rates tend to decrease it. Therefore, by controlling the redox potential during fermentation using the above method, the actual redox potential of the fermentation broth will fluctuate between the upper and lower limits of the set redox potential range.

[0017] By simultaneously adjusting both the stirring speed and the air flow rate, the oxidation-reduction potential can be regulated. On the one hand, the air flow rate can be reduced, and the upward trend of the oxidation-reduction potential can be promoted by increasing the stirring speed. On the other hand, by combining the increase of stirring speed with the air flow rate, the introduced air can be fully utilized, allowing the introduced air to dissolve better in the fermentation broth. This reduces the required air flow rate and also reduces the generation of a large number of bubbles in the fermentation broth due to the introduction of a large amount of air, thus avoiding contamination of the fermentation broth.

[0018] Optionally, the stirring speed ranges from 300 to 900 rpm, the aeration rate ranges from 1.0 to 2.5 vvm, the initial stirring speed is 300 rpm, and the initial aeration rate is 1.0 vvm. Selecting these stirring speed and aeration rate ranges avoids damage to the bacteria due to excessively high stirring speeds, which could affect the growth and spore production of Bacillus subtilis. It also avoids contamination of the fermentation broth due to excessive aeration rates causing excessive bubble formation. By synergistically combining these appropriate aeration rate and stirring speed, the aforementioned side effects can be mitigated, while also facilitating the adjustment of the redox potential of Bacillus subtilis during the fermentation process.

[0019] Optionally, in the process of controlling the redox potential of the fermentation broth: when the redox potential is lower than the lower limit of the redox potential range, the stirring speed of the fermentation broth is increased first, and then the air flow rate is increased; when the redox potential is higher than the upper limit of the redox potential range, the air flow rate is decreased first, and then the stirring speed of the fermentation broth is decreased.

[0020] When it is necessary to increase the redox potential, first increase the stirring speed of the fermentation broth, and then increase the air flow rate. This ensures that the introduced air can dissolve quickly into the fermentation broth, reducing the generation of bubbles. Similarly, when it is necessary to decrease the redox potential of the fermentation broth, first decrease the air flow rate, and then decrease the stirring speed of the fermentation broth. This also reduces the generation of bubbles. Since the fermentation process takes a long time, it may be necessary to adjust the air flow rate and stirring speed multiple times. Therefore, by reducing the amount of bubbles generated during the fermentation process, the safety of the fermentation process can be improved, and contamination of the fermentation broth can be avoided.

[0021] Optionally, the fermentation broth contains 47-53 g / L soy protein isolate, 72-78 g / L malt syrup, 4-8 g / L calcium chloride, 2-4 g / L dipotassium hydrogen phosphate, 2-6 g / L magnesium sulfate heptahydrate, 0.4-0.6 g / L zinc sulfate heptahydrate, 0.5-1.5 g / L manganese sulfate monohydrate, and 0.01-0.03 g / L riboflavin.

[0022] Optionally, after the fermentation process is completed, the spore rate is not less than 90%.

[0023] The beneficial effects of this application include, but are not limited to:

[0024] 1. In the growth and spore production of Bacillus subtilis, the spore quantity can be increased by controlling the redox potential. Furthermore, by dividing the fermentation process into a first fermentation stage and a second fermentation stage, maintaining a high potential in the first fermentation stage and a low potential in the second fermentation stage, the spore quantity can be effectively increased, the spore rate can be improved, the carbon source conversion rate can be increased, and the air intake can be significantly reduced.

[0025] 2. By optimizing and selecting the high potential in the first fermentation stage and the low potential in the second fermentation stage, the amount of air used can be further reduced, thereby lowering costs and increasing carbon source conversion rate, which in turn increases spore yield.

[0026] 3. By adjusting both the air flow rate and the fermentation broth stirring speed, the redox potential of the fermentation broth was adjusted, and a suitable adjustment range was further optimized and selected. On the one hand, stirring the fermentation broth allows the introduced air to dissolve more fully, reducing the generation of bubbles and thus preventing contamination. Stirring also reduces the air flow rate, saving costs. On the other hand, avoiding excessively high stirring speeds minimizes damage to the Bacillus subtilis growth cells.

[0027] 4. By further optimizing the order of adjusting the stirring speed and air flow rate of the fermentation broth, specifically, when it is necessary to increase the redox potential, the stirring speed should be increased first and then the air flow rate should be increased; when it is necessary to decrease the redox potential, the air flow rate should be decreased first and then the stirring speed should be decreased. This can reduce the amount of bubbles generated in the fermentation broth during fermentation, avoid contamination of the fermentation broth, and thus ensure the spore yield of Bacillus subtilis. Detailed Implementation

[0028] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0029] Unless otherwise specified, the raw materials and catalysts used in the embodiments of this application were all purchased commercially.

[0030] The Bacillus subtilis used in this application is designated GDMCC 1.372 and was purchased from the Guangdong Provincial Microbial Culture Collection Center (GDMCC).

[0031] Example 1

[0032] Strain activation

[0033] LB solid medium was used and the pH was adjusted to 7.25. The culture was sterilized at 121℃ for 30 min and then put into use. For activation, the inoculum was streaked on LB solid medium and then incubated at 37℃ for 32 h. The LB solid medium contained 10 g / L sodium chloride, 10 g / L peptone and 5 g / L yeast extract.

[0034] Secondary activation of bacterial strains

[0035] LB solid medium was used and the pH was adjusted to 7.25. The culture was sterilized at 121℃ for 30 min and then put into use. For activation, the inoculum was spread on LB solid medium as a slant and then incubated at 37℃ for 56 h. The LB solid medium contained 10 g / L sodium chloride, 10 g / L peptone and 5 g / L yeast extract.

[0036] Seed culture

[0037] Liquid culture medium A was used, containing 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, 10 g / L glycerol, and 0.01 g / L riboflavin, with the pH adjusted to 6.8. One loopful of the cultured slant culture was inoculated into an Erlenmeyer flask containing liquid culture medium A. The shaker speed was set to 200 rpm. OD 600 The optical density absorbance value was controlled between 4 and 6, and the cells were incubated at 37°C for 7.5 hours in a constant temperature incubator.

[0038] Fermentation production

[0039] Liquid culture medium B was used, which contained 50 g / L soy protein isolate, 75 g / L malt syrup, 6 g / L calcium chloride, 3 g / L dipotassium hydrogen phosphate, 4 g / L magnesium sulfate heptahydrate, 0.5 g / L zinc sulfate heptahydrate, 1 g / L manganese sulfate monohydrate, and 0.02 g / L riboflavin. The pH was adjusted to 6.6, the inoculum size was 10%, the dissolved oxygen (DO) content was controlled to be no less than 40% during fermentation, the fermentation temperature was 36℃, and the fermentation time was 32 h.

[0040] During fermentation, the redox potential is controlled between 50 and 350 mV. The initial aeration rate is set to 1.0 vvm (air volume / culture volume / min, i.e., the ratio of aeration per minute to the actual liquid volume in the tank), and the initial stirring speed is 300 rpm. Then, the initial redox potential in the fermenter is measured using a redox electrode, and the potential regulation process is initiated: when the redox potential detection value is lower than the lower limit of the set range, the stirring speed and air aeration rate are increased; when the redox potential detection value is higher than the upper limit of the set range, the air aeration rate and stirring speed are decreased. The air aeration rate is controlled between 1.0 and 2.5 vvm, and the stirring speed is controlled between 300 and 900 rpm.

[0041] Example 2

[0042] The difference from Example 1 is that the total fermentation time is 32 hours, and the fermentation process is divided into two stages. In the first fermentation stage, the redox potential of the fermenter is controlled at 150-350 mV, and the fermentation time is 16 hours. After the first fermentation stage, the second fermentation stage is carried out. In the second fermentation stage, the redox potential of the fermenter is controlled at 50-150 mV, and the fermentation time is 16 hours. In both fermentation stages, the fermentation temperature is set at 39°C, and other conditions are the same as in Example 1.

[0043] Example 3

[0044] The difference from Example 2 is that the redox potential of the fermenter is controlled at 50-150mV in the first fermentation stage and at 150-350mV in the second fermentation stage, while the other conditions are the same as in Example 2.

[0045] Example 4

[0046] The difference from Example 2 is that the redox potential of the fermenter is controlled at 245-255 mV in the first fermentation stage and at 95-105 mV in the second fermentation stage. All other conditions are the same as in Example 2.

[0047] Example 5

[0048] The difference from Example 2 is that the fermentation time of the first fermentation stage is 14 hours and the fermentation time of the second fermentation stage is 18 hours, while all other conditions are the same as in Example 2.

[0049] Example 6

[0050] The difference from Example 2 is that the fermentation time of the first fermentation stage is 18 hours and the fermentation time of the second fermentation stage is 14 hours, while all other conditions are the same as in Example 2.

[0051] Example 7

[0052] The difference from Example 2 is that the total fermentation time is 28 hours, the fermentation time of the first fermentation stage is 14 hours, the fermentation time of the second fermentation stage is 14 hours, and all other conditions are the same as in Example 2.

[0053] Example 8

[0054] The difference from Example 2 is that the total fermentation time is 40 hours, the fermentation time of the first fermentation stage is 20 hours, the fermentation time of the second fermentation stage is 20 hours, and all other conditions are the same as in Example 2.

[0055] Example 9

[0056] The difference from Example 2 is that the fermentation process in this example is set at a temperature of 33°C, while all other conditions are the same as in Example 2.

[0057] Example 10

[0058] The difference from Example 2 is that during the fermentation process, the air flow rate is controlled between 1.0 and 2.0 vvm, the stirring speed is between 300 and 1200 rpm, and all other conditions are the same as in Example 2.

[0059] Comparative Example 1

[0060] The difference from Example 1 is that, during the fermentation process, the redox potential of the fermentation broth is not controlled, only the DO value is controlled to be no less than 10%, and all other conditions are the same as in Example 1.

[0061] Comparative Example 2

[0062] The difference from Example 1 is that, during the fermentation process, the redox potential of the fermentation broth is controlled between 350 and 450 mV.

[0063] Test Example 1

[0064] The spore count, carbon source conversion rate, spore count, and air usage of Examples 1-10 and Comparative Examples 1-2 after fermentation were tested and statistically compared. The specific results are shown in Table 1 below.

[0065] Table 1

[0066]

[0067]

[0068] Analysis of Table 1 shows that, compared with Comparative Example 1, in the growth and spore production of Bacillus subtilis, controlling the redox potential between 50 and 350 mV significantly improved spore quantity and carbon source conversion rate compared to not controlling the redox potential during fermentation. Compared with Example 1, Example 2, by dividing the fermentation process into two stages—controlling the potential at a high potential of 150–350 mV in the first fermentation stage and at a low potential of 50–150 mV in the second fermentation stage—not only achieved very high spore quantity and carbon source conversion rate, but also a spore rate exceeding 90%, while reducing air consumption and saving resources. It can increase spore yield, showing significant improvement; compared with Example 2, Example 3 shows that the first fermentation stage is controlled at a low potential of 50-150mV, and the second fermentation stage is controlled at a high potential of 150-350mV. Although the spore yield is increased compared with Comparative Example 1 without controlling the redox potential, the improvement is smaller than that of Example 2; compared with Example 2, Example 4 shows that by further stabilizing the potential fluctuation range in the two stages, the potential in the first fermentation stage is controlled at 245-255mV, and the potential in the second fermentation stage is controlled at 95-105mV. Compared with Example 2, it can further reduce the amount of air used and increase the spore yield.

[0069] Comparing Examples 2, 5, and 6, it can be seen that if the fermentation time of the first fermentation stage is too short, although the spore rate is high, the biomass of Bacillus subtilis after growth in the first fermentation stage will be too low, and the reduction in the initial number of Bacillus subtilis will ultimately lead to a significant decrease in spore yield. If the fermentation time of the first fermentation stage is too long, the potential will remain high for a long time in the first fermentation stage, resulting in a low biomass of Bacillus subtilis after growth in the first fermentation stage, and the final spore rate will also be low due to the short time of the second fermentation stage. Comparing Examples 2, 7, and 8, it can be seen that if the total fermentation time is too short, the growth of Bacillus subtilis and the spore production process will be affected by the short time, resulting in a low final spore yield. Both carbon source conversion rate and spore rate are too low. When the total fermentation cycle is too long, although the spore quantity, carbon source conversion rate and spore rate will increase, the air ventilation will increase significantly, resulting in a significant increase in cost. Compared with Example 2, Example 9 shows that when the fermentation temperature is too low during the fermentation process, it will affect the growth of Bacillus subtilis and the spore production process, resulting in a significant increase in air ventilation, and the spore quantity, carbon source conversion rate and spore rate will also decrease significantly. Compared with Example 2, Example 10 shows that if the stirring speed is increased too much in order to reduce air usage, the high speed will damage the cells during fermentation production. Although the air usage is reduced, it will lead to a decrease in spore yield.

[0070] Compared with Comparative Example 2, if the control potential range is too high, the growth cycle of Bacillus subtilis will be too short, the number of spores will be reduced, and the overall production efficiency and carbon source conversion rate will be reduced. In addition, in order to maintain a high redox potential, the air flow rate will be increased, the air utilization rate will be low, and excessive ventilation will easily lead to excessive foam in the fermenter, which will make it easy to be contaminated. This will lead to the addition of too much defoamer, resulting in excessive toxicity, which is not conducive to the growth and spore formation of Bacillus subtilis.

[0071] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A method for regulating Bacillus subtilis growth for spore production using a redox electrode, characterized by, In the fermentation production process, a first fermentation stage and a second fermentation stage are included, the redox potential of the fermentation broth in the first fermentation stage is controlled in the range of 150-350 mV, the redox potential of the fermentation broth in the second fermentation stage is controlled in the range of 50-150 mV, the ratio of the fermentation time in the first fermentation stage to the fermentation time in the second fermentation stage is 1:0.8-1.2, the total fermentation time of the first fermentation stage and the second fermentation stage is 30-38 h, and the temperature of the fermentation broth is 36-39℃; The method for controlling the redox potential of the fermentation broth is as follows: (1) determining the redox potential of the fermentation broth, setting the initial stirring speed and the initial air input amount; (2) when the redox potential is lower than the lower limit of the redox potential range, increasing the stirring speed and the air input amount of the fermentation broth, and when the redox potential is higher than the upper limit of the redox potential range, decreasing the stirring speed and the air input amount of the fermentation broth.

2. The method of claim 1, wherein, The redox potential of the fermentation broth in the first fermentation stage is controlled in the range of 245-255 mV, and the redox potential of the fermentation broth in the second fermentation stage is controlled in the range of 95-105 mV.

3. The method of claim 1, wherein, The stirring speed is in the range of 300-900 rpm, and the air input amount is in the range of 1.0-2.5vvm.

4. The method of claim 1, wherein, In the process of controlling the redox potential of the fermentation broth: when the redox potential is lower than the lower limit of the redox potential range, the stirring speed of the fermentation broth is first increased, and then the air input amount is increased; when the redox potential is higher than the upper limit of the redox potential range, the air input amount is first decreased, and then the stirring speed of the fermentation broth is decreased.

5. The method of claim 1, wherein, The fermentation broth includes soybean protein isolate 47-53 g / L, malt syrup 72-78 g / L, calcium chloride 4-8 g / L, dipotassium hydrogen phosphate 2-4 g / L, magnesium sulfate heptahydrate 2-6 g / L, zinc sulfate heptahydrate 0.4-0.6 g / L, manganese sulfate monohydrate 0.5-1.5 g / L, and riboflavin 0.01-0.03 g / L.

6. The method of claim 1, wherein, After the fermentation production is completed, the spore rate is not less than 90%.

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