A bacillus subtilis strain producing polyglutamic acid and its application in soil remediation of northeast agroforestry-grassland complex system

Abstract

This invention relates to a polyglutamic acid-producing Bacillus subtilis strain and its application in soil remediation in agroforestry-grassland integrated systems in Northeast China. The taxonomic name of Bacillus subtilis is... Bacillus subtilis The depositary institution for this invention is the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 32822 and deposit date of November 27, 2024. This invention isolates and screens highly efficient, low-temperature polyglutamic acid-producing *Bacillus subtilis* from soils in different agroforestry-grassland integrated systems in Northeast China. It grows well at 10-20℃ and secretes polyglutamic acid. Its application in Northeast agroforestry-grassland integrated systems has a significant impact on the vegetative growth and economic traits of maize, reducing stress effects, increasing crop absorption of available nutrients from the soil, and thus increasing maize yield. After application, it can effectively improve soil physicochemical properties, increase seed germination rate, and enhance seedling resistance.

Description

Technical Field

[0001] This invention relates to a polyglutamic acid-producing Bacillus subtilis strain and its application in soil remediation in agroforestry-grassland integrated systems in Northeast China, belonging to the field of microbiology. Background Technology

[0002] With the continuous increase in population and the scarcity of arable land and water resources, how to utilize the different types of soil under the agroforestry-grassland composite system in Northeast China is an urgent problem to be solved. For soil under farmland shelterbelts, the common practice is to dig trenches and cut roots, increasing the area of ​​farmland occupied. This severely restricts farmers' enthusiasm for creating and protecting shelterbelts, leading to a certain degree of degradation in many areas. Common methods for soil improvement include deep plowing and topsoil application, physicochemical methods, and agricultural plant improvement methods. These can all improve soil and promote plant development to a certain extent, but they are costly and prone to secondary pollution. Soil remediation microbial agents are a relatively new type of improvement product that can more effectively and comprehensively develop and utilize obstacle soils. Many experts and scholars recognize the improvement effect of microorganisms. They are not only low-cost but also have the advantages of no secondary pollution. In northern my country, the temperature is low during the crop sowing season, and microorganisms at room temperature survive and reproduce slowly under cold climate conditions, which can easily have adverse effects on spring sowing. Therefore, finding a microorganism that can promote the efficient absorption of soil nutrients by crops under low-temperature conditions is of great significance. Based on their optimal growth temperature, low-temperature microorganisms are classified into two categories: psychrophiles and psychrophiles. Psychrophiles can grow and reproduce at an optimal temperature of around 15°C, and can survive at around 20°C. Psychrophiles, on the other hand, can survive at around 0°C, with an optimal temperature of around 20°C for growth and reproduction. The biosphere contains abundant microbial resources, including low-temperature microorganisms. Therefore, fully utilizing these resources in daily life and production is crucial. Low-temperature microorganisms can generally grow and reproduce at room temperature, requiring no additional energy, making them readily available and widely distributed. Consequently, research on low-temperature microorganisms has become a focus, attracting the attention of experts and scholars.

[0003] To date, polyglutamic acid secreted by microorganisms has good water retention properties. Polyglutamic acid is a high molecular weight amino acid polymer with a large number of free carboxyl groups on its molecular chain. It has good water solubility and strong water retention properties. At the same time, it has excellent properties such as non-toxicity, biodegradability, and low production cost. It has shown very broad application prospects in agriculture. However, there are very few reports on Bacillus subtilis that produces polyglutamic acid at low temperatures.

[0004] During the crop sowing season in northern my country, temperatures are low, and microorganisms at room temperature survive and reproduce slowly in cold climates, which can negatively impact spring sowing. Therefore, finding a microorganism that can promote crop rooting under low-temperature conditions is of great significance.

[0005] Furthermore, the types of polyglutamic acid-producing strains currently being studied are limited, and many of them suffer from drawbacks such as high production costs, insufficient and unstable polyglutamic acid production capacity, and narrow pH adaptation range. Therefore, it is necessary to continue to explore and develop new strains with high activity and a wider range of effects to produce polyglutamic acid at low temperatures. This has greater advantages and potential in terms of energy saving and cost reduction, and is of great development prospect.

[0006] In summary, there are no reports on a polyglutamic acid-producing Bacillus subtilis strain and its application in soil remediation of agroforestry-grassland integrated systems in Northeast China. Summary of the Invention

[0007] The purpose of this invention is to address the above-mentioned problems by providing a polyglutamic acid-producing Bacillus subtilis strain and its application in soil remediation in agroforestry-grassland integrated systems in Northeast China.

[0008] To achieve the above-mentioned objectives of the present invention, the present invention adopts the following technical solution: A strain of Bacillus subtilis, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32822, on November 27, 2024.

[0009] The 16S rDNA sequence of Bacillus subtilis (ZY136) is as follows:

[0010] The biological characteristics of Bacillus subtilis are as follows: on PDA medium, it is smooth and moist with regular edges and a convex, opaque center; the colony color is pale yellow, glossy, and spherical and emulsifiable; the mucus has a distinct string-pulling effect when picked up, exhibiting typical colony characteristics of polyglutamic acid-producing bacteria.

[0011] Method for isolating the bacterial strain: Soil suspensions collected from different regions of Northeast China's agroforestry-grassland composite system were inoculated at a 10% inoculum into an inorganic salt medium containing 10 g / L glutamic acid. After culturing for 3 days at 15°C with a shaker at 150 r / min, the inoculum was transferred to an inorganic salt medium with a glutamic acid concentration of 15 g / L at 15°C and cultured for another 3 days. This process was repeated 3 times, with the glutamic acid concentrations in the inorganic salt medium used for the three cycles being 20 g / L, 25 g / L, and 30 g / L, respectively. The culture was then plated and incubated in a constant temperature incubator at 15°C for 72 hours. Single colonies were picked and transferred to an inorganic salt medium with a glutamic acid concentration of 30 g / L to obtain the initial screening bacterial suspension.

[0012] Inorganic salt (seed) culture medium supplements are composed of the following raw materials by weight: NH4+ ４ Cl is 7g, K2HPO ４ The concentrations of the culture medium are as follows: 1g of CaCl2, 0.5g of MgSO4, 0.05g of MnSO4, 0.02g of FeCl3, 13.5g of citric acid, 10-30g of L-glutamic acid, and 950g of distilled water; the pH of the culture medium is 4.8-7.2.

[0013] Strain screening: a. Initial screening: The selected strains were inoculated into seed culture medium and cultured at 15℃ and 150 r / min for 20 h. The apparent viscosity of the fermentation broth was measured with a viscometer, and the strains with higher viscosity were selected as the initial screening strains. b. Secondary screening: The amount of polyglutamic acid in the fermentation broth was determined using the ninhydrin colorimetric method; Pretreatment of fermentation broth: The fermentation broth was centrifuged at 5000 r / min for 15 min, and 5 mL of the supernatant was taken. Then, 5 mL of 6 mol / L HCl solution was added to a sterile colorimetric tube and hydrolyzed at 110℃ for 24 h. After hydrolysis, the pH was adjusted to 7 with 6 mol / L NaOH solution and diluted to 200 mL in a volumetric flask. Take 1 mL of the above hydrolyzed sample into a sterile colorimetric tube, add 1 mL of ninhydrin solution, 1 mL of phosphate buffer, and distilled water to a final volume of 6 mL. Heat in a 100°C water bath for 20 min, then cool to room temperature and bring the volume to 25 mL. Measure the absorbance at 570 nm using a UV spectrophotometer. Calculate the glutamic acid content after hydrolysis using the glutamic acid standard curve. Repeat the above steps with the unhydrolyzed supernatant to calculate the glutamic acid content in the unhydrolyzed fermentation broth. Subtract the two values ​​to obtain the polyglutamic acid yield in the fermentation broth. Determine its polyglutamic acid production capacity and perform re-selection.

[0014] Pot-based field trials were conducted to determine the effectiveness of the strain in actual production.

[0015] The advantages of this invention are: 1. Beneficial effects of the present invention: Due to strain ZY136 ( Bacillus subtilis ZY136 was obtained by screening from soil in a Northeast agroforestry-grassland composite system at 15℃. After multiple subcultures and enrichment cultures at 15℃, the strain was preserved to a greater extent. Bacillus subtilis The stable, low-temperature production of polyglutamic acid provides a natural microbial resource for soil remediation under agroforestry-grassland integrated systems in Northeast China, offering a material basis and technical support for the subsequent development of microbial agents.

[0016] 2. The fertilizer product produced by this invention has the unique characteristic of producing polyglutamic acid at low temperature, and is inexpensive. After application, it can effectively improve the physical and chemical properties of the soil, increase the seed germination rate, and enhance the stress resistance of seedlings. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to the embodiments.

[0018] A polyglutamic acid-producing Bacillus subtilis strain and its application in soil remediation in agroforestry-grassland integrated systems in Northeast China. The Bacillus subtilis culture was deposited at the China General Microbiological Culture Collection Center, China. Bacillus subtilis (ZY136) has the accession number CGMCC No.32822 and the accession date is November 27, 2024.

[0019] Methods for isolating the strains: Soil suspensions collected from multiple locations in Northeast China's agroforestry-grassland composite system were inoculated at a 10% inoculum into an inorganic salt medium containing 10 g / L glutamic acid. After culturing for 3 days at 10°C with a shaker at 150 rpm, the inoculum was transferred at 5°C to an inorganic salt medium with a glutamic acid concentration of 15 g / L and cultured for another 3 days. This process was repeated three times, with the glutamic acid concentrations in the inorganic salt medium being 20 g / L, 25 g / L, and 30 g / L, respectively. The samples were then plated and incubated at 5°C for 72 hours. Single colonies were picked and transferred to an inorganic salt medium with a glutamic acid concentration of 30 g / L to obtain the initial screening bacterial suspension.

[0020] Inorganic salt (seed) culture medium supplements are composed of the following raw materials by weight: NH4+ ４ Cl is 7g, K2HPO ４ The concentrations of the culture medium are as follows: 1g of CaCl2, 0.5g of MgSO4, 0.05g of MnSO4, 0.02g of FeCl3, 13.5g of citric acid, 10-30g of L-glutamic acid, and 950g of distilled water; the pH of the culture medium is 4.8-7.2.

[0021] Strain screening: a. Initial screening: The selected strains were inoculated into seed culture medium and cultured at 37℃ and 150 r / min for 20 h. The apparent viscosity of the fermentation broth was measured with a viscometer, and the strains with higher viscosity were selected as the initial screening strains.

[0022] b. Secondary screening: The yield of polyglutamic acid in the fermentation broth was determined by the ninhydrin colorimetric method.

[0023] Pretreatment of fermentation broth: The fermentation broth was centrifuged at 5000 r / min for 15 min, and 5 mL of the supernatant was taken. Then, 5 mL of 6 mol / L HCl solution was added to a sterile colorimetric tube and hydrolyzed at 110℃ for 24 h. After hydrolysis, the pH was adjusted to 7 with 6 mol / L NaOH solution and diluted to 200 mL in a volumetric flask.

[0024] Take 1 mL of the above hydrolyzed sample into a sterile colorimetric tube, add 1 mL of ninhydrin solution, 1 mL of phosphate buffer, and distilled water to a final volume of 6 mL. Heat in a 100°C water bath for 20 min, then cool to room temperature and bring the volume to 25 mL. Measure the absorbance at 570 nm using a UV spectrophotometer. Calculate the glutamic acid content after hydrolysis using the glutamic acid standard curve. Repeat the above steps with the unhydrolyzed supernatant to calculate the glutamic acid content in the unhydrolyzed fermentation broth. Subtract the two values ​​to obtain the polyglutamic acid yield in the fermentation broth. Determine its polyglutamic acid production capacity and perform reselection.

[0025] This invention employs a plate culture method to isolate and purify strains from three different soil samples, initially screening out 140 rapidly growing bacterial strains. Strains capable of forming filaments were selected, and after shake-flask fermentation and further screening, 12 strains with high apparent viscosity were obtained. Following three subcultures and shake-flask measurements, strain ZY136 showed the highest viscosity (115 mPa·s), stable polyglutamic acid production capacity, and a yield as high as 1.846 g / L.

[0026] Morphological characteristics: On PDA medium, strain ZY136 is smooth and moist with regular edges and a centrally convex, opaque ridge. Colonies are pale yellow, glossy, and spherical with a milky appearance. The mucus exhibits a distinct stringy effect when picked up, characteristic of polyglutamic acid-producing bacteria. Gram-positive. Under a microscope, the bacteria appear as long rods with capsules and spores originating from the center.

[0027] Molecular biological identification of strain ZY136 was performed: Genomic DNA was extracted from the culture of strain ZY136 after 7 days of incubation in PDA medium at 15℃, and 16S rDNA was amplified by PCR. The PCR products were subjected to agarose gel electrophoresis, and the samples were sent to Shanghai Panoson Biotechnology Co., Ltd. for 16S rDNA gene sequencing. The sequencing results of strain ZY136 16S rDNA were compared with the NCBI database using BLAST. The results showed that strain ZY136 had more than 95% homology with Bacillus subtilis strain SBMP4. Strains with high sequence similarity were selected, and a phylogenetic tree was constructed using MEGA 7.0. The results showed that strain ZY136 and Bacillus subtilis strain SBMP4 were homologous, and the phylogenetic distance was 94. Therefore, strain ZY136 belongs to the genus Bacillus subtilis. Bacillus subtilis ).

[0028] Case 1: The experimental site was a slightly saline soil near a shelterbelt. The soil type was brown soil, with moderate fertility and good irrigation and drainage conditions. Topsoil samples were taken for testing before the experiment; the basic physical and chemical properties of the soil are shown in Table 1. The test crop was maize, variety Zhengdan 958.

[0029] Table 1. Basic physical and chemical properties of soil

[0030] Experimental design: The experiment consisted of 3 treatments, with a plot area of ​​50 m²; the experiment was repeated 3 times in a randomized block design.

[0031] Treatment 1: Conventional fertilization + test soil remediation microbial agent (Bacillus subtilis ZY136).

[0032] Treatment 2: Conventional fertilization + equal amount of inactivation substrate.

[0033] Treatment 3: Conventional fertilization.

[0034] Experimental method: Treatment 1 was to apply soil remediation microbial agent as a base fertilizer on the basis of conventional fertilization, at a rate of 100 kg per 667 m².

[0035] Treatment 2 involved applying the same amount of inactivated substrate as Treatment 1 as basal application, in addition to conventional fertilization.

[0036] Treatment 3 involved conventional fertilization: 40 kg / 667 m² of compound fertilizer (20-10-10) was applied at corn planting time, and 20 kg / 667 m² of urea was applied as a top dressing at the corn tasseling stage.

[0037] Experimental requirements: During the experiment, no other fertilizers were applied except as required by the experimental plan, and other management measures remained the same; each harvest was conducted separately for each plot, with weighing, yield calculation, and observation and recording of crop growth and economic traits.

[0038] Field management: Soil remediation microbial agent was applied as a base fertilizer on May 15, 2021, and the plots were divided for sowing. On September 25, 2021, the economic traits of corn were investigated in each plot. Corn was harvested and yield was measured in October.

[0039] Experimental results and analysis: 1. Effects of different treatments on soil physicochemical properties.

[0040] Table 2 shows the interannual variations in the soil physicochemical properties of the experimental site. In treatment 1, soil pH, salinity, and bulk density decreased by 3.79%, 5.71%, and 2.89%, respectively, while soil organic matter increased by 5.92%. In summary, the application of soil remediation microbial agents effectively improved soil salinity, rapidly modified the topsoil, and fully utilized soil functions, providing strong support for soil ecosystem restoration and crop yield increase.

[0041] Table 2. Changes in soil physicochemical properties under different treatments

[0042] 2. Effects of different treatments on vegetative growth and economic traits of maize.

[0043] Table 3 shows that the application of soil remediation microbial agents has a significant impact on the vegetative growth and economic traits of maize. It can alleviate leaf senescence, increase ear length, and reduce tip barrenness. Specifically, maize plant height, ear length, and ear diameter all increased. Treatment 1 showed increases in plant height, ear length, and ear diameter of 0.87%, 1.76%, and 1.31% compared to treatment 3, respectively. Therefore, soil remediation microbial agents promote the vegetative growth of maize, resulting in better economic traits.

[0044] Table 3. Effects of different treatments on physiological traits of maize

[0045] 3. The impact of different treatments on maize yield.

[0046] Table 4 shows that the yield of the soil remediation microbial agent treatment increased by 5.53% compared with conventional fertilization. To further verify the differences between the soil remediation microbial agent treatment, the substrate treatment, and conventional fertilization of maize, the maize yield results were analyzed using SPSS 19.0 software for difference testing (Table 5), and multiple comparisons were performed using the Ducan method (Table 6). The analysis of variance results showed that the soil remediation microbial agent had a statistically significant effect on maize yield, and the difference reached a highly significant level. The multiple analysis results showed that the yield of treatment 1 was significantly different from that of treatments 2 and 3, while the yield difference between treatments 2 and 3 was not significant.

[0047] Table 4. Corn Yield Statistics

[0048] Table 5. Analysis of variance of maize plot yield

[0049] Table 6. Duncan's Multiple Comparisons

[0050] Conclusion: After applying soil remediation microbial agents, the soil is reasonably improved with prolonged application time, the topsoil is rapidly modified, and soil functions are fully utilized, providing strong support for soil ecosystem restoration and crop yield increase. The application of soil remediation microbial agents has a significant impact on maize vegetative growth and economic traits, alleviating leaf senescence, increasing ear length, reducing tip barrenness, and thus increasing maize yield.

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