Bacillus subtilis for producing gamma-polyglutamic acid by open-type fermentation of sweet potato starch wastewater and application of bacillus subtilis

Abstract

The invention provides bacillus subtilis for producing gamma-polyglutamic acid by open fermentation of sweet potato starch wastewater and application of the bacillus subtilis. The preservation number of the bacillus subtilis 1109 is CGMCC (China General Microbiological Culture Collection Center) No.32748; the preservation date is November 22, 2024; the preservation unit is China General Microbiological Culture Collection Center (CGMCC). According to the bacillus subtilis for producing the gamma-polyglutamic acid through open fermentation of the sweet potato starch wastewater, the sweet potato starch wastewater is subjected to open fermentation, the gamma-polyglutamic acid slow-release liquid fertilizer is produced, and the yield of the gamma-polyglutamic acid in fermentation liquor can reach 10-20 g / L.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to Bacillus subtilis for producing gamma-polyglutamic acid by open fermentation of sweet potato starch wastewater and its application. Background Art

[0002] With the continuous advancement of industrial technology, the field of agricultural food deep processing has developed rapidly. However, this development is also accompanied by environmental pollution problems, especially the problem of wastewater discharge generated during food processing, which has become a focus of widespread attention. Taking sweet potato starch processing as an example, its wastewater usually contains high chemical oxygen demand (COD) and biological oxygen demand (BOD). If it is directly discharged without effective treatment, it may cause water pollution, soil pollution and environmental sanitation problems, thereby affecting the ecological balance and human health.

[0003] At present, traditional wastewater treatment methods mainly include physical methods, chemical methods and anaerobic biological fermentation methods. Although these methods can reduce the pollution burden to a certain extent, their treatment costs are high and they may cause secondary pollution problems, which will bring huge operational pressure to food companies. In addition, these methods generally fail to achieve resource recovery and reuse, so they have certain limitations in promoting the sustainable development of the circular economy. Summary of the invention

[0004] In view of this, the present invention aims to overcome the defects in the prior art and proposes a Bacillus subtilis for producing γ-polyglutamic acid by open fermentation of sweet potato starch wastewater and its application.

[0005] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] The present invention provides Bacillus subtilis 1109 for producing γ-polyglutamic acid by open fermentation of sweet potato starch wastewater, wherein the deposit number of the Bacillus subtilis 1109 is: CGMCC No. 32748; the deposit date is November 22, 2024; the deposit unit is the General Microbiological Center of the China Microbiological Culture Collection Administration. The deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the taxonomic name of the Bacillus subtilis 1109 is Bacillus subtilis.

[0007] The present invention also provides an open fermentation method of Bacillus subtilis 1109 using sweet potato starch wastewater, comprising the following steps:

[0008] (1) inoculating glycerol bacteria into a culture medium to obtain an activated strain;

[0009] (2) inoculating the activated strain into a seed liquid culture medium to obtain a seed liquid after culturing, inoculating the obtained seed liquid into a slow-release liquid fertilizer fermentation medium, and performing open fermentation.

[0010] Furthermore, the temperature of the culture step in step (2) is 30-45°C and the time is 10-20 hours.

[0011] Furthermore, the seed liquid culture medium of the culturing step of step (2) comprises the following components in mass percentage: 0.5-2.0% (w / v) glucose, 0.5-2% (w / v) peptone, 0.1-1% (w / v) yeast extract, 0.5-15% (w / v) sodium chloride, and 0.5-1.5% (w / v) sodium glutamate; the pH value of the seed liquid culture medium of the culturing step of step (2) is 5.0-8.0.

[0012] Furthermore, the inoculation amount of the seed solution in step (2) is 10-30% (v / v).

[0013] Furthermore, the fermentation temperature of the open fermentation step in step (2) is 30-47° C., the rotation speed is 300-800 rpm, and the fermentation time is 10-24 hours.

[0014] Furthermore, the slow-release liquid fertilizer fermentation medium in step (2) comprises the following components in mass percentage: 80-100% (v / v) sweet potato starch wastewater, 0.8-1.5% (w / v) yeast powder, 0.02-0.2% (w / v) ferric sulfate, 0.01-0.2% (w / v) sodium dihydrogen phosphate, 0.01-0.2% (w / v) disodium hydrogen phosphate, 0.01-0.3% (w / v) magnesium sulfate, 0.01-0.1% (w / v) calcium chloride, and 0.8%-1.5% (w / v) sodium glutamate; the pH value of the slow-release liquid fertilizer fermentation medium in step (2) is 5.0-8.0.

[0015] Furthermore, the fermentation method also includes a continuous fermentation step. After the open fermentation is completed, 70-90% of the material is discharged, 10-30% of the bacterial liquid is retained, and fresh slow-release liquid fertilizer fermentation culture medium is added to carry out continuous fermentation for 10-20 batches.

[0016] The present invention also provides an application of the Bacillus subtilis 1109, which is an application of the Bacillus subtilis 1109 in preparing liquid fertilizer; the liquid fertilizer is a gamma-polyglutamic acid slow-release liquid fertilizer; and the liquid fertilizer is prepared by fermenting sweet potato starch wastewater.

[0017] Furthermore, the liquid fertilizer is used for planting peppers or tomatoes. The slow-release liquid fertilizer is used as a microbial fertilizer to improve soil fertility.

[0018] Sweet potato starch wastewater is rich in organic matter, such as starch, crude fat, free amino acids and crude fiber, and has certain nutritional value. In recent years, the use of bioconversion technology for wastewater treatment and resource recovery has gradually become a promising technical route. In particular, the use of Bacillus (Bacillus) that can produce broad-spectrum antimicrobial peptides as the core strain, combined with open fermentation technology, converts the organic matter in sweet potato starch wastewater into slow-release liquid fertilizer, which not only effectively reduces the environmental pollution of wastewater, but also can achieve efficient utilization of wastewater resources and provide high-quality fertilizer resources for agricultural production. This method not only provides an innovative solution to the problem of wastewater discharge, but also opens up a new path for the sustainable development of the agricultural economy.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] The Bacillus subtilis of the present invention for producing γ-polyglutamic acid by open fermentation of sweet potato starch wastewater does not require sterilization treatment, and can efficiently utilize organic matter (such as starch, crude fat, free amino acids, etc.) in sweet potato starch wastewater as a carbon source to produce high value-added slow-release liquid fertilizer (γ-polyglutamic acid). Compared with traditional wastewater treatment methods, the technology of the present invention can significantly reduce wastewater treatment costs and realize resource utilization of wastewater; the Bacillus subtilis strain can efficiently produce slow-release liquid fertilizer in 10 to 20 rounds of continuous fermentation, and can be applied to the planting of crops such as small peppers, tomatoes, etc., which helps to improve crop growth quality and soil fertility.

[0021] The Bacillus subtilis for producing gamma-polyglutamic acid by open fermentation of sweet potato starch wastewater of the present invention produces gamma-polyglutamic acid slow-release liquid fertilizer by open fermentation of sweet potato starch wastewater, and the yield of gamma-polyglutamic acid in the fermentation liquid can reach 10-20 g / L. DETAILED DESCRIPTION

[0022] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0023] The basic parameters of the sweet potato starch wastewater used in the embodiment of the present invention are shown in Table 1.

[0024] Table 1 Basic parameters of sweet potato starch wastewater

[0025]

[0026]

[0027] The present invention will be described in detail below with reference to the embodiments.

[0028] Example 1 Production of slow-release liquid fertilizer (γ-polyglutamic acid) by fermentation of sweet potato starch wastewater

[0029] The original strain was purchased from Shangcheng Beina Chuanglian Biotechnology Co., Ltd., Bacillus subtilis BNCC189983, and Bacillus subtilis 1109 was obtained by ARTP mutagenesis screening. It was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on November 22, 2024, with a deposit number of CGMCC No.32748, and the address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The following content uses this strain as the production strain. Bacillus subtilis BNCC190341 was used as a control strain, which was purchased from Shangcheng Beina Chuanglian Biotechnology Co., Ltd.

[0030] Bacillus subtilis 1109 has the properties in Table 2.

[0031] Table 2 Morphological characteristics and physiological and biochemical characteristics of colonies

[0032] Experimental Project result Experimental Project result Cell shape Rod VP test + Gram stain + Indole production test － Spore + Urease test + Anaerobic growth + Oxidase test + Growth at 50℃ + Casein test + 10% NaCl growth + Cellulose hydrolysis test + Nitrate reduction test + Starch hydrolysis test + Citrate utilization test + Gelatin liquefaction test +

[0033] Starch wastewater was collected, and the Bacillus subtilis plate was activated and inoculated into the seed liquid culture medium, the specific ingredients of which were as follows: 1% (w / v) glucose, 0.5% (w / v) peptone, 1% (w / v) yeast extract, 1% (w / v) sodium chloride, 0.5% (w / v) sodium glutamate, and the pH value was 7.2. After overnight culture, the mixture was inoculated into a 7L fermentation tank. The fermentation medium formula was: 100% (v / v) starch wastewater, 1.5% (w / v) yeast powder, 0.02% (w / v) ferric sulfate, 0.01% (w / v) sodium dihydrogen phosphate, 0.01% (w / v) disodium hydrogen phosphate, 0.02% (w / v) magnesium sulfate, 0.02% (w / v) calcium chloride, 1.5% (w / v) sodium glutamate) for fermentation culture. No sterilization was required for open fermentation. The culture speed was 600 rpm, the ventilation ratio was 2 vvm, the temperature was 37 ° C, and the fermentation cycle was 14 h. The culture process of Bacillus subtilis 1109 and BNCC190341 was the same as above. As shown in Table 3, Bacillus subtilis 1109 showed better growth performance. Compared with BNCC190341, the growth rate was faster and the biomass was higher in the sweet potato starch wastewater fermentation medium.

[0034] Table 3 Growth comparison of Bacillus subtilis starch wastewater fermentation medium

[0035] Time (h) Bacillus subtilis 1109 BNCC190341 0 1.51±0.65 1.57±0.45 2 4.67±0.26 2.87±0.21 4 8.52±0.55 4.32±0.89 6 12.67±1.21 6.44±0.55 8 14.89±1.30 8.29±0.74 10 18.65±0.56 10.13±1.01 12 17.22±0.48 12.44±0.58 14 17.87±0.77 12.65±0.98

[0036] Example 2 Experiment on the Growth Promotion of Peppers by Slow-release Liquid Fertilizer

[0037] Starch wastewater was collected, and the Bacillus subtilis plate was activated and inoculated into the seed liquid culture medium, the specific ingredients of which were as follows: 1% (w / v) glucose, 0.5% (w / v) peptone, 1% (w / v) yeast extract, 1% (w / v) sodium chloride, 0.5% (w / v) sodium glutamate, and the pH value was 7.2. After overnight culture, the mixture was inoculated into a 7L fermentation tank. The fermentation medium formula was: 100% (v / v) starch wastewater, 1.5% (w / v) yeast powder, 0.02% (w / v) ferric sulfate, 0.01% (w / v) sodium dihydrogen phosphate, 0.01% (w / v) disodium hydrogen phosphate, 0.02% (w / v) magnesium sulfate, 0.02% (w / v) calcium chloride, 1.5% (w / v) sodium glutamate). No sterilization was required for open fermentation. The culture speed was 600 rpm, the ventilation ratio was 2 vvm, the temperature was 37°C, and the fermentation cycle was 14 hours. The culture process of Bacillus subtilis1109 and BNCC190341 was the same as above. After centrifugation, the supernatant containing slow-release liquid fertilizer was diluted 200 times for liquid fertilizer application experiments. The peppers were sprayed from the seedling stage to the initial flowering stage. A total of 3 experimental groups were designed, with pure water control as CK, application of Stanley compound fertilizer as EC-1, application of Stanley compound fertilizer + BNCC190341 slow-release liquid fertilizer spraying as EC-2, and application of Stanley compound fertilizer + Bacillus subtilis 1109 slow-release liquid fertilizer spraying as EC-3. As shown in Figure 4, the use of Stanley compound fertilizer + Bacillus subtilis slow-release liquid fertilizer has a good improvement on the agronomic shape of small peppers, especially the use of Stanley compound fertilizer + Bacillus subtilis1109 slow-release liquid fertilizer spraying has the most prominent improvement on the agronomic traits of small peppers.

[0038] Table 4 Effects of spraying slow-release liquid fertilizer on agronomic traits of peppers

[0039] Application method Plant height / cm Effective branches / Number of fruits per plant Fruit weight / g CK 64.8 5.3 98.4 35.2 EC-1 66.2 6.4 115.2 40.7 EC-2 67.6 6.5 121.7 44.6 EC-3 70.16 7.1 125.4 47.8

[0040] Example 3 Effect of Bacillus subtilis on tomato growth and yield

[0041] Starch wastewater was collected, and the Bacillus subtilis plate was activated and inoculated into the seed liquid culture medium, the specific ingredients of which were as follows: 1% (w / v) glucose, 0.5% (w / v) peptone, 1% (w / v) yeast extract, 1% (w / v) sodium chloride, 0.5% (w / v) sodium glutamate, and the pH value was 7.2. After overnight culture, the mixture was inoculated into a 7L fermenter with a fermentation medium formula of: 100% (v / v) starch wastewater, 1.5% (w / v) yeast powder, 0.02% (w / v) ferric sulfate, 0.01% (w / v) sodium dihydrogen phosphate, 0.01% (w / v) sodium hydrogen phosphate, 0.02% (w / v) magnesium sulfate, 0.02% (w / v) calcium chloride, and 1.5% (w / v) sodium glutamate. The fermentation was carried out in an open fermentation tank without sterilization. The culture speed was 600 rpm, the ventilation ratio was 2 vvm, the temperature was 37°C, and the fermentation cycle was 14 h. The culture process of Bacillus subtilis1109 and BNCC190341 was the same as above. After centrifugation, the supernatant containing slow-release liquid fertilizer was diluted 200 times for liquid fertilizer application experiment. The spraying period of tomato fruit foliar fertilizer was once in the seedling stage, once in the young fruit stage, and twice in the fruit expansion stage. A total of three groups of experiments were designed, with pure water control as CK, application of Stanley compound fertilizer as EC-1, application of Stanley compound fertilizer + BNCC190341 slow-release liquid fertilizer as EC-2, and application of Stanley compound fertilizer + Bacillus subtilis 1109 slow-release liquid fertilizer as EC-3. As shown in Figure 5, the use of Stanley compound fertilizer + Bacillus subtilis slow-release liquid fertilizer has a good improvement on tomato yield, especially the use of Stanley compound fertilizer + Bacillus subtilis1109 slow-release liquid fertilizer spraying has the most prominent improvement on the agronomic traits of tomatoes.

[0042] Table 5 Effect of slow-release liquid fertilizer on tomato yield

[0043] deal with Plant height / cm Stem diameter / mm Leaf length / cm Leaf width / cm Number of fruits per plant CK 147.03±7.45 11.92±0.37 20.57±1.03 10.98±0.87 16.13±1.72 EC-1 152.61±5.72 12.43±0.21 21.77±0.29 11.39±0.34 17.22±0.81 EC-2 153.69±8.21 12.56±0.33 22.46±0.77 12.03±0.17 18.44±1.12 EC-3 154.69±7.79 13.45±0.29 24.58±0.56 13.03±0.55 20.13±0.65

[0044] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Bacillus subtilis 1109 for producing γ-polyglutamic acid by open fermentation of sweet potato starch wastewater, characterized in that: The deposit number of the Bacillus subtilis 1109 is: CGMCC No.32748; the deposit date is November 22, 2024; and the deposit unit is the General Microbiology Center of the China Microbiological Culture Collection Administration.

2. An open fermentation method using sweet potato starch wastewater using Bacillus subtilis 1109 according to claim 1, characterized in that: The steps include: (1) inoculating glycerol bacteria into a culture medium to obtain an activated strain; (2) inoculating the activated strain into a seed liquid culture medium to obtain a seed liquid after culturing, inoculating the obtained seed liquid into a slow-release liquid fertilizer fermentation medium, and performing open fermentation.

3. The open fermentation method utilizing sweet potato starch wastewater according to claim 2, characterized in that: The temperature of the culture step in step (2) is 30-45°C and the time is 10-20h.

4. The open fermentation method utilizing sweet potato starch wastewater according to claim 2, characterized in that: The seed liquid culture medium of the culturing step of step (2) comprises the following components in mass percentage: 0.5-2.0% (w / v) glucose, 0.5-2% (w / v) peptone, 0.1-1% (w / v) yeast extract, 0.5-15% (w / v) sodium chloride, and 0.5-1.5% (w / v) sodium glutamate; the pH value of the seed liquid culture medium of the culturing step of step (2) is 5.0-8.

0.

5. The open fermentation method using sweet potato starch wastewater according to claim 2, characterized in that: The inoculation amount of the seed solution in step (2) is 10-30% (v / v).

6. The open fermentation method using sweet potato starch wastewater according to claim 2, characterized in that: The fermentation temperature of the open fermentation step in step (2) is 30-47°C, the rotation speed is 300-800rpm, and the time is 10-24 hours.

7. The open fermentation method using sweet potato starch wastewater according to claim 2, characterized in that: The slow-release liquid fertilizer fermentation medium in step (2) comprises the following components in percentage by mass: 80-100% (v / v) sweet potato starch wastewater, 0.8-1.5% (w / v) yeast powder, 0.02-0.2% (w / v) ferric sulfate, 0.01-0.2% (w / v) sodium dihydrogen phosphate, 0.01-0.2% (w / v) disodium hydrogen phosphate, 0.01-0.3% (w / v) magnesium sulfate, 0.01-0.1% (w / v) calcium chloride, and 0.8%-1.5% (w / v) sodium glutamate; the pH value of the slow-release liquid fertilizer fermentation medium in step (2) is 5.0-8.

0.

8. The open fermentation method using sweet potato starch wastewater according to claim 2, characterized in that: The fermentation method also includes a continuous fermentation step. After the open fermentation is completed, 70-90% of the material is discharged, 10-30% of the bacterial liquid is retained, and fresh slow-release liquid fertilizer fermentation medium is added to carry out continuous fermentation for 10-20 batches.

9. The use of Bacillus subtilis 1109 according to claim 1, characterized in that: The application of the Bacillus subtilis 1109 in the preparation of liquid fertilizer; the liquid fertilizer is gamma-polyglutamic acid slow-release liquid fertilizer; the liquid fertilizer is prepared by fermenting sweet potato starch wastewater.

10. The use of Bacillus subtilis 1109 according to claim 9, characterized in that: The liquid fertilizer is used for planting peppers or tomatoes.

Images