A strain of Bacillus licheniformis with high production of β-mannanase and its application

Abstract

The present invention belongs to the field of microbial technology, and particularly relates to a bacillus mutant strain of a high-yield alkaline mannanase and its application technology. The strain is specifically Bacillus licheniformis (Bacillus licheniformis) GAN2-10, with a deposit number of CGMCC NO.26080. The mannanase enzyme activity produced by the fermentation of the mutant strain reaches 35980U / mL, which is more than 40% higher than the mannanase enzyme activity produced by the original strain. The optimum reaction temperature of the obtained mannanase is 40°C, and a good enzyme degradation effect can be exerted in a low temperature environment, and it has good heat resistance, and after being incubated for 4h at 80°C, the relative enzyme activity is more than 80%. In addition, the enzyme has an optimum reaction pH of 9.5, has good alkali resistance, and after being incubated for 4h at pH 11.0, the relative enzyme activity is more than 85%. Due to the good enzymatic properties of the above-mentioned enzyme, it can exert a good enzymolysis effect in a low temperature and alkaline environment, so it can be widely used in industries such as decolorization and bleaching.

Description

Technical field:

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a mutant strain of Bacillus licheniformis with high beta-mannanase production and industrial application technology thereof. Background technology:

[0002] β-Mannanase, as one of the new enzyme preparations, is a hemicellulase that can degrade mannans to produce small soluble mannose or manno-oligosaccharides.

[0003] β-mannanase from different sources has different characteristics. For this reason, the enzyme has been widely used in various industries. For example, in the feed industry, the degradation of mannan by β-mannanase to produce manno-oligosaccharides can improve the intestinal flora of livestock and poultry, reduce harmful bacterial infections, improve the health of livestock and poultry, and promote the growth of livestock and poultry. In the baking industry, the addition of β-mannanase can enhance gluten tension, improve dough elasticity, increase the mechanical bearing capacity of dough, and increase the volume of bread. In the beer industry, the enzyme can remove dissolved oxygen from beer, prevent beer from being oxidized and deteriorated, and extend the shelf life of beer. Similarly, in the dairy industry, the use of β-mannanase can prevent the fat in milk from being oxidized, inhibit the growth of miscellaneous bacteria, and ensure the quality of milk. In addition, β-mannanase is also used in other industrial fields such as medicine, cosmetics, tobacco, petroleum, papermaking, and textiles.

[0004] β-mannanase, as a textile enzyme, has been publicly studied as early as the end of the last century. It can be used as an enzyme preparation for ramie bio-degumming. After degumming, the strength of ramie fiber is enhanced and the fiber quality is greatly improved. In addition, β-mannanase can also be used for dye decolorization and bleaching in the textile, washing and papermaking industries. As a major dye producer, my country's annual dye exports account for about 1 / 5 of the world trade. With high-yield dyes, a large amount of industrial wastewater is discharged, which brings huge production and governance pressure to the dye production industry and environmental protection departments. The new research and development technology of alkaline β-mannanase can alleviate some of the pressure in the industry to a certain extent. β-mannanase, as an environmentally friendly renewable resource-biological enzyme preparation, has the advantages of a wide range of substrates, non-toxicity, no pollution, mild action conditions, and simple and controllable production process. Under the current international background of rising raw material prices and scarce resources, it is imperative to find a process technology with high production capacity, low energy consumption, fast efficiency and low cost.

[0005] This study used composite mutagenesis technology to transform the original strain, and used shake flask screening to selectively screen strains with significantly improved enzyme activity and stable genetic traits. Under optimized fermentation process conditions, the production capacity of the high-yield mutant strain was greatly improved, and the yield of alkaline β-mannanase was increased by more than 40%. Summary of the invention:

[0006] The present invention relates to a mutant strain with high alkaline β-mannanase production and its application in industrial fermentation. To a certain extent, it solves the practical problems of low production and high production cost of existing β-mannanase, which is of great significance for improving efficiency and reducing costs of feed enzyme preparation manufacturers, and has far-reaching significance for improving efficiency and reducing emissions in the textile dye industry.

[0007] One of the technical solutions provided by the present invention is a Bacillus with high production of alkaline β-mannanase, specifically Bacillus licheniformis GAN2-10. The strain GAN2-10 was obtained by plasma-nitrosoguanidine composite mutagenesis screening of Bacillus licheniformis strain G-1. It has been deposited in the General Microbiology Center of China Microorganism Collection Committee on November 8, 2022, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a deposit number of CGMCC NO.26080.

[0008] The second technical solution provided by the present invention is the use of Bacillus licheniformis GAN2-10 in the production of alkaline β-mannanase, especially the production of alkaline β-mannanase by liquid fermentation. The specific technical solution is as follows:

[0009] 1. Seed solution preparation: Inoculate a single colony of GAN2-10 into 300 mL of culture medium (LB medium) for cultivation;

[0010] Culture conditions: temperature 30℃, rotation speed 220r / min, culture time 36h.

[0011] 2. Seed tank culture: inoculate the seed solution into the seed tank culture medium at an inoculation rate of 3-5%;

[0012] Culture conditions: temperature 30-32℃, stirring speed 250-300r / min, ventilation volume 1.0-1.5vvm, tank pressure 0.06-0.08MPa, culture for 20h.

[0013] 3. Fermentation tank culture: inoculate the seed solution into the fermentation tank culture medium at an inoculation rate of 6-8% for cultivation;

[0014] Culture conditions: temperature 30-32°C, stirring speed 250-300r / min, ventilation volume 1.0-1.5vvm, tank pressure 0.06-0.08MPa, pH 8.7-9.0 (ammonia is added for adjustment when the pH is lower than 8.5, and feed is added for adjustment when the pH is higher than 9.2), the dissolved oxygen content is controlled at 15-20% in the initial fermentation stage (0-50h), and the dissolved oxygen is controlled at 25-30% after 50h. When the enzyme activity does not increase significantly after 100-110h of fermentation and the bacteria are severely autolyzed, the tank is released.

[0015] 4. Fed-batch culture: Start feeding when the pH is higher than 9.2 and control the pH in the range of 8.7-9.0.

[0016] 5. The culture medium involved in the above culture process is as follows:

[0017] LB medium: 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast powder, and the rest is deionized water, pH 8.7-9.0;

[0018] Seed tank culture medium: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, the rest is deionized water, pH 8.7-9.0;

[0019] Fermentation tank culture medium: konjac flour 15-20g / L, soybean meal powder 20-23g / L, soy peptone 18-20g / L, diammonium phosphate 10-12g / L, magnesium sulfate heptahydrate 12-15g / L, potassium dihydrogen phosphate 15-18g / L, the rest is water, pH 8.7-9.0;

[0020] Feed medium: glucose powder 300-500g / L, corn steep liquor 8-10g / L, diammonium phosphate 1.0-1.3g / L, magnesium sulfate heptahydrate 1.0-1.3g / L, potassium dihydrogen phosphate 1.3-1.5g / L, the rest is water, pH 8.7-9.0.

[0021] After 100-110 hours of fermentation culture, the β-mannanase activity in the fermentation broth can reach 34000-35900 U / mL.

[0022] The alkaline β-mannanase prepared by the above fermentation method has the following enzymatic properties:

[0023] 1. The optimum reaction temperature is 40°C, and the action temperature range is wide;

[0024] 2. The optimum reaction pH is 9.5, and the action pH range is wide;

[0025] 3. Good heat resistance, keeping the enzyme activity at 80℃ for 4 hours, and the enzyme activity retention rate is more than 80%;

[0026] 4. Good alkali resistance. When stored at pH 11.0 for 4 hours, the enzyme activity retention rate is more than 85%.

[0027] Beneficial effects:

[0028] 1. The present invention carries out plasma-nitrosoguanidine composite mutagenesis screening on the original strain, and obtains a beneficial mutant strain Bacillus licheniformis GAN2-10 with a fermentation level improved by 40% and stable inheritance.

[0029] 2. The present invention provides a liquid industrial fermentation technology for high-yield β-mannanase. The production of β-mannanase by this technology has higher fermentation activity, lower production cost, and better enzymatic properties, which greatly improves the utilization efficiency of raw materials and has great significance for enterprises to reduce costs and increase efficiency.

[0030] 3. The β-mannanase produced by the mutagenic strain Bacillus licheniformis GAN2-10 provided by the present invention has greatly improved enzyme activity, good enzyme activity release performance under low temperature and alkaline environment, and excellent heat resistance, and can exert a good enzymatic hydrolysis effect. Description of the drawings:

[0031] Figure 1 Enzyme activity curve of β-mannanase at different reaction temperatures;

[0032] Figure 2 Temperature stability curve of β-mannanase;

[0033] Figure 3 Enzyme activity curve of β-mannanase at different reaction pH;

[0034] Figure 4 pH stability curve of β-mannanase. Specific implementation method:

[0035] The present invention is further explained below through specific implementation schemes. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the implementation schemes should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these implementation schemes, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0036] Example 1 Strain mutagenesis screening

[0037] 1. Plasma mutagenesis:

[0038] (1) Take a fresh slant of the recently preserved Bacillus licheniformis strain G-1, scoop out an appropriate amount of bacteria and culture it in a seed bottle for 36 hours at 30°C and 220 r / min. Stop the culture when the bacterial concentration reaches 0.55-0.6 (OD 600nm). Use the bacterial solution at this time as the mutagenic bacterial suspension.

[0039] (2) Turn on the normal temperature and pressure plasma mutagenesis system and turn on the ultraviolet lamp for sterilization. After sterilization, take 10 μL of bacterial suspension on the slide, transfer the slide to the operating table for mutagenesis, and set the mutagenesis time to 30 s, 45 s, 60 s, 90 s, 120 s, 150 s, and 180 s respectively.

[0040] (3) After the mutagenesis is completed, the mutagenesis solution is quickly diluted in a gradient manner and spread on the screening plate. It is cultured in a 30°C incubator for 30 hours and the colonies are finally observed and counted.

[0041] (4) 15-20 single colonies with good growth conditions were selected from each mutagenesis group for subculture and shake flask culture. (5) After repeated shake flask verification, the strain GA2-17 with the largest increase in enzyme activity and stable inheritance was finally selected as the starting strain for re-mutation.

[0042] 2. Nitrosoguanidine mutagenesis:

[0043] (1) Take a fresh slant of the recently preserved strain GA2-17, scoop out an appropriate amount of bacteria and culture it in a seed bottle for 36 hours at 30°C and 220 r / min. Stop the culture when the bacterial concentration reaches 0.55-0.6 (OD600 nm).

[0044] (2) The above seed solution was centrifuged, the supernatant was discarded, and the obtained bacteria were resuspended in 5 ml of Tris buffer. 1 mL of the resuspended solution was taken and placed in a conical flask, and Tris buffer and nitrosoguanidine mutagen were added. The solution was shaken on a shaker at 30°C and 220 r / min for 25 min. After treatment, the solution was centrifuged, the supernatant was discarded, and the solution was washed several times. A certain concentration of glycerol was added to the obtained bacteria, and the solution was resuspended and stored in aliquots. At the same time, the mutagenic solution was diluted to different gradients and spread on the screening plate for 30 h. The culture conditions were 30°C and 220 r / min.

[0045] (3) According to the changes in colony morphology, 15-20 single colonies with good growth conditions were selected for subculture and shake flask culture. Finally, a strain GAN2-10 with significantly improved enzyme activity was obtained. Its genetic traits were stable, and the enzyme activity in shake flask fermentation was nearly 90% higher than that of the starting strain. The β-mannanase produced by this strain has a wide range of action temperature and pH, and has good heat and alkali resistance.

[0046] 3. Culture medium:

[0047] The culture medium involved in the above-mentioned composite mutagenesis screening process is as follows:

[0048] (1) Screening culture medium: 5 g / L konjac flour, 6 g / L yeast powder, 0.6 g / L magnesium sulfate heptahydrate, 0.9 g / L potassium dihydrogen phosphate, 0.5 g / L calcium chloride, 13 g / L agar, and the rest is deionized water, pH 8.8-9.0;

[0049] (2) Seed flask culture medium: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, and the rest is deionized water, pH 8.8-9.0;

[0050] (3) Shake flask culture medium: 9 g / L konjac flour, 8 g / L yeast extract, 0.7 g / L magnesium sulfate heptahydrate, 1.1 g / L potassium dihydrogen phosphate, 0.6 g / L calcium chloride, and the rest is deionized water, pH 8.8-9.0.

[0051] 4. Subculture experiment of high-yield β-mannanase strain GAN2-10:

[0052] The starting strain G-1 and the high-yield strain GAN2-10 were streaked and cultured on the screening plates. Single colonies with good growth were selected and placed in seed bottles, and cultured at 30°C and 220 r / min for 36 h, and then fermented in shake flasks. Finally, the enzyme activity of the fermentation liquid in the shake flask was determined. The strain was subcultured continuously, and the experimental results are shown in Table 1.

[0053] Table 1. Results of β-mannanase activity test of strain GAN2-10 in shake flasks

[0054]

[0055] The results in Table 1 show that the mutant strain GAN2-10 was subcultured for 6 generations, and the enzyme activity was stable, which was increased by more than 90% compared with the starting strain, indicating that the mutant strain had good genetic stability and could be used in industrial production fermentation.

[0056] Example 2 Method for determining β-mannanase activity of the present invention

[0057] 1. Definition of enzyme activity:

[0058] Under certain conditions (unless otherwise specified, the conditions are 40°C and pH 9.0), the amount of enzyme required for 1 g or 1 ml of enzyme to release 1 μmol of reducing sugar (mannose) per minute is one enzyme activity unit U.

[0059] 2. Drawing of standard curve:

[0060] Weigh 0.5000 g of anhydrous mannose, add 0.1 mol / L sodium acetate buffer (pH 9.0) to dissolve it, and make up to 50 mL.

[0061] 0 mL, 0.50 mL, 1.00 mL, 1.50 mL, 2.00 mL, 2.50 mL, and 3.00 mL of mannose solution were respectively taken and made up to 50 mL with 0.1 mol / L sodium acetate buffer (pH 9.0) to prepare mannose standard solutions with concentrations of 0 mg / mL, 0.10 mg / mL, 0.20 mg / mL, 0.30 mg / mL, 0.40 mg / mL, 0.50 mg / mL, and 0.60 mg / mL.

[0062] Take 1.00mL of each of the above concentration series of mannose standard solutions and add them to the colorimetric tube, then add 1.00mL of sodium acetate buffer and 2.5mL of DNS reagent. Shake evenly and heat in a boiling water bath for 5min. Cool quickly and dilute to 12.5mL with distilled water. Zero with the standard blank sample and measure the absorbance at 540nm. Draw a standard curve with mannose concentration as the y-axis and absorbance as the x-axis.

[0063] 3. Enzyme activity determination method:

[0064] 1.00 mL of appropriately diluted enzyme solution was pipetted into three colorimetric test tubes respectively and placed in a 40°C water bath for equilibration for 10 min. At the same time, 5.0 mL of 0.6% locust bean gum solution substrate (0.1 mol / L pH 9.0 sodium acetate buffer) was pipetted into another test tube and placed at 40°C for equilibration for 10 min.

[0065] Add 2.5 mL of DNS reagent to the colorimetric tube with enzyme solution, shake evenly, add 1.0 mL of the preheated substrate solution, and keep warm at 40°C for 30 min. After the insulation is completed, heat in a boiling water bath for 5 min, cool rapidly, add water to make up to 12.5 mL, and shake evenly. This is used as a blank control.

[0066] In the other two colorimetric test tubes with enzyme solution added, add 1.0 mL of the preheated substrate solution respectively, shake evenly, and keep warm at 40°C for 30 min. After the insulation is completed, immediately add 2.5 mL of DNS reagent, shake evenly, heat in a boiling water bath for 5 min, cool rapidly, add water to make up to 12.5 mL, shake evenly, and measure the absorbance at 540 nm.

[0067] 4. Calculation formula of mannanase activity:

[0068]

[0069] Where:

[0070] X------mannanase enzyme activity, U / mL;

[0071] A------absorbance value;

[0072] K-------The slope of the standard curve;

[0073] B------The intercept of the standard curve;

[0074] M-------The molar mass of mannose, M(C6HO6)=180.16g / mol;

[0075] t-------enzyme reaction time, min;

[0076] n------the dilution multiple of the sample;

[0077] 1000----Conversion factor, 1mmol=1000μmol.

[0078] Example 3 Production of mannanase by liquid fermentation of strain GAN2-10

[0079] 1. Seed solution preparation: Inoculate a single colony of GAN2-10 into 300 mL of culture medium (LB medium) for cultivation. Culture conditions: temperature 30°C, speed 220 r / min, culture time 36 h;

[0080] 2. Seed tank culture: inoculate the seed solution into the seed tank culture medium at a 4% inoculation rate. Culture conditions: temperature 30°C, stirring speed 260r / min, ventilation volume 1.0vvm, tank pressure 0.06MPa, culture for 20h;

[0081] 3. Fermentation tank culture: Inoculate the seed solution into the fermentation tank culture medium at an inoculation rate of 7% for culture. Culture conditions: temperature 30°C, stirring speed 260r / min, ventilation volume 1.2vvm, tank pressure 0.06MPa, pH 8.8 (add ammonia to adjust when pH is lower than 8.5, and add feed to adjust when pH is higher than 9.2), control the dissolved oxygen content at 16% in the initial fermentation period (0-50h), and control the dissolved oxygen content at 26% after 50h. Ferment to 100-110h, if the enzyme activity does not increase significantly and the bacteria are seriously autolyzed, release the tank;

[0082] 4. Fed-batch culture: start feeding when the pH is higher than 9.2 and control the pH in the range of 8.7-9.0;

[0083] 5. The culture medium involved in the above culture process is as follows:

[0084] LB medium: 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast powder, and the rest is deionized water, pH 9.0;

[0085] Seed tank culture medium: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, the rest is deionized water, pH 9.0;

[0086] Fermentation tank medium: 16 g / L konjac flour, 20 g / L soybean meal, 18 g / L soy peptone, 10 g / L diammonium phosphate, 12 g / L magnesium sulfate heptahydrate, 16 g / L potassium dihydrogen phosphate, and the rest is water, pH 9.0;

[0087] Feed medium: glucose powder 400 g / L, corn steep liquor 8 g / L, diammonium phosphate 1.2 g / L, magnesium sulfate heptahydrate 1.0 g / L, potassium dihydrogen phosphate 1.3 g / L, the rest is water, pH 9.0.

[0088] 6. Liquid fermentation test results of strain GAN2-10:

[0089] Bacillus licheniformis strain GAN2-10 was used for liquid fermentation in a fermenter (30 L fermenter), and the fermentation enzyme activity is shown in the following table.

[0090] Table 2.30L small tank fermentation test results

[0091]

[0092]

[0093] The fermentation experiment was carried out for 6 batches, and the enzyme activity of the fermentation broth reached more than 34000U / ml, which was more than 40% higher than that of the original strain. After 110h of fermentation culture, the β-mannanase activity in the fermentation broth could reach up to 35980U / mL. This shows that the strain GAN2-10 has improved and stable mannanase production ability, that is, the mutant strain has good stable genetic performance and can be used for industrial fermentation production.

[0094] Example 4 Enzymatic properties of β-mannanase

[0095] 1. Optimum reaction temperature:

[0096] The supernatant of the fermentation broth obtained from the fermentation of the third batch of GAN2-10 in Example 3 was used as the mannanase sample. Under the condition of pH 9.0, the enzyme activity at different reaction temperatures was measured. The temperatures were 25, 30, 35, 40, 45, 50, 55, 60, 70, and 75°C. After the measurement, the relative enzyme activity at each temperature was calculated (the enzyme activity at 40°C was calculated as 100%). Figure 1As shown, the optimum reaction temperature of the enzyme is 40°C, the enzyme activity is above 85% at 25°C, and above 80% at 75°C. This indicates that the mannanase produced by the mutant strain GAN2-10 has a wide range of action temperatures and can exert its good degradation effect at room temperature, indicating that the enzyme is suitable for use in the textile and papermaking industries.

[0097] 2. Temperature resistance:

[0098] The supernatant of the fermentation broth obtained from the fermentation of the third batch of GAN2-10 in Example 3 was used as a mannanase sample, and was placed at different temperatures, which were set to 50, 55, 60, 65, 70, 75, 80, and 85° C., respectively, for 4 hours, and then the enzyme activity was measured and the relative enzyme activity was calculated (the untreated enzyme activity was calculated as 100%). Figure 2 It can be seen that after being kept at 80℃ for 4 hours, the relative enzyme activity was above 80%, and when the temperature rose to 85℃, the relative enzyme activity was still above 75%. The results showed that the mannanase produced by the mutant strain had strong heat resistance.

[0099] 3. Optimum reaction pH:

[0100] The supernatant of the fermentation broth obtained by fermenting the third batch of GAN2-10 in Example 3 was used as a mannanase sample. The mannanase activity was measured at pH values ​​of 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, and 11.0 at 40° C., and the relative enzyme activity was calculated (with the highest enzyme activity as 100%). The measurement results are as follows: Figure 3 The results showed that the optimum reaction pH of mannanase was 9.5, and the relative enzyme activity at pH 10.5 was still above 90%, which indicated that the mannanase produced by the mutant strain was suitable for application in alkaline environments, such as in the textile and papermaking industries.

[0101] 4. Alkali resistance:

[0102] The supernatants of the fermentation broths obtained from the fermentation of the third batch of GAN2-10 and G-1 in Example 3 were used as mannanase samples, respectively. They were placed in different pH environments, with pH values ​​of 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, and 11.0, respectively, and incubated for 4 hours. The enzyme activity was then determined and the relative enzyme activity was calculated (with the untreated enzyme activity as 100%). The results are shown in FIG. Figure 4 After incubation at pH 10.5 for 4 hours, the relative enzyme activity was above 90%. Compared with the original strain, the alkali resistance of the mannanase produced by the mutant strain GAN2-10 was greatly enhanced, which was more conducive to the enzyme's enzymatic effect in an alkaline environment.

[0103] The above-mentioned embodiments only express several implementation methods of the present invention, and the contents described are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that for ordinary technicians in this field, the above-mentioned embodiments can also make several deformations, combinations and improvements without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A Bacillus licheniformis strain producing alkaline mannanase, characterized in that: The strain is specifically Bacillus licheniformis ( Bacillus licheniformis )GAN2-10, accession number CGMCC NO.26080.

2. The Bacillus licheniformis according to claim 1 ( Bacillus licheniformis )The application of GAN2-10 is characterized by: It is used in the fermentation production of mannanase.

3. The use according to claim 2, characterized in that The method for producing alkaline mannanase by liquid fermentation is as follows: According to the inoculation amount of 6-8%, the seed liquid was transferred to the fermentation medium and cultured in the fermenter. The culture conditions were as follows: temperature 30-32℃, stirring speed 250-300 r / min, ventilation volume 1.0-1.5 vvm, tank pressure 0.06-0.08 MPa, pH 8.7-9.

0. When the pH was lower than 8.5, ammonia was added for adjustment. When the pH was higher than 9.2, feed was added for adjustment. At the initial fermentation time of 0-50 h, the dissolved oxygen content was controlled at 15-20%. After 50 h, the dissolved oxygen was controlled at 25-30%. When the enzyme activity did not increase significantly after 100-110 h of fermentation and the bacteria were severely autolyzed, the tank was released.

4. The use according to claim 3, characterized in that: The fermentation tank culture medium is composed of: 15-20 g / L konjac flour, 20-23 g / L soybean meal, 18-20 g / L soybean peptone, 10-12 g / L diammonium phosphate, 12-15 g / L magnesium sulfate heptahydrate, 15-18 g / L potassium dihydrogen phosphate, and the rest is water, pH 8.7-9.0; Feed medium: glucose powder 300-500 g / L, corn steep liquor 8-10 g / L, diammonium phosphate 1.0-1.3 g / L, magnesium sulfate heptahydrate 1.0-1.3 g / L, potassium dihydrogen phosphate 1.3-1.5 g / L, the rest is water, pH 8.7-9.0.

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