Sinorhizobium meliloti H9 as well as high-density fermentation process and application thereof

Abstract

The invention provides sinorhizobium meliloti H9 as well as a high-density fermentation process and application thereof, and belongs to the technical field of microorganisms. The sinorhizobium meliloti is classified and named as sinorhizobium meliloti, and the sinorhizobium meliloti is preserved in the China General Microbiological Culture Collection Center on September 9, 2024, and the preservation number of the sinorhizobium meliloti is CGMCC (China General Microbiological Culture Collection Center) No. 31900. The sinorhizobium meliloti H9 provided by the invention is a high-efficiency phosphate-solubilizing bacterium and has strong stress resistance, and the sinorhizobium meliloti H9 and the high-density fermentation process thereof provided by the invention are beneficial to developing a novel efficient, broad-spectrum, green and environment-friendly phosphate-solubilizing bacterium biological agent.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to Sinorhizobium alfalfa H9 and a high-density fermentation process and application thereof. Background Art

[0002] Phosphorus (P) is essential for plant growth and is a limiting factor for crop yields. The total phosphorus content in cultivated land worldwide is high, and the available phosphorus content in plants is low, which seriously affects plant growth and agricultural development. The long-term use of phosphorus fertilizers will affect soil fertility, lead to the accumulation of harmful elements in the soil, and pollute the agricultural ecosystem. Therefore, the effective use of phosphorus resources is crucial to promoting the quality and yield of crops.

[0003] Microorganisms are an important natural resource for developing "green methods" and can be applied to agriculture as a green technology. In addition to chemical phosphate fertilizers, the use of microbial phosphate solubilization and mineralization seems to be the only possible way to increase the efficiency of plant phosphorus absorption. It is known that there are many microorganisms in the soil and rhizosphere that can dissolve phosphorus, called phosphate-solubilizing microorganisms (PSM), which play an important role in phosphorus cycle processes such as organic phosphorus mineralization, insoluble inorganic phosphorus solubilization and phosphorus absorption. These microorganisms have the advantages of being environmentally friendly, low cost and high biological efficiency. They have now become the focus of attention and are considered to be part of the "fresh" green revolution. However, there are few studies on Sinorhizobium alfalfa, which mainly focus on the degradation of fungal toxins by composite or single microbial agents, such as patents CN202210058217.2, CN202310244939.1, CN202210058221.9, CN202211557631.4; as a biocatalyst, such as patents CN202010580723.9, CN202110424332.2; and the preparation of vitamin B 12 Applications in, such as patents CN202010012124.7, CN202010289270.4, CN202010012114.3, CN202010012144.4, CN202010012125.1.

[0004] Sinorhizobium meliloti has been widely used in the fields of biotechnology, chemical industry, medicine, resources and environment, but its application in promoting growth by dissolving phosphorus is limited to the application of Sinorhizobium meliloti XGL026 described in patent CN201710303203.1 in enhancing the growth of Xinjiang alfalfa plants. At present, there is still a lack of research on using Sinorhizobium meliloti as a specific growth-promoting strain resource for high-density fermentation. Summary of the invention

[0005] In order to solve the deficiencies in the prior art, the purpose of the present invention is to provide a Sinorhizobium alfalfa H9 and its high-density fermentation process and application. The present invention isolates and screens a Sinorhizobium alfalfa H9 with strong phosphate solubility and stress resistance, explores the effects of different culture conditions on the biomass of strain H9, and obtains the optimal culture process of Sinorhizobium alfalfa H9, providing a theoretical basis and experimental basis for further preparing PSB bacterial agents, improving soil performance and increasing crop yields.

[0006] In order to achieve the above object, the specific scheme adopted by the present invention is:

[0007] In the first aspect, the present invention provides a kind of Sinorhizobium meliloti H9, which is classified and named Sinorhizobium meliloti, and has been deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee on September 9, 2024, with the deposit number of CGMCC No.31900.

[0008] In a second aspect, the present invention provides a high-density fermentation process of the above-mentioned Sinorhizobium meliloti H9, wherein the above-mentioned Sinorhizobium meliloti H9 is inoculated into a fermentation medium for fermentation culture; The fermentation medium contains: 48.32 g / L lactose, 11.32 g / L yeast powder, and 3.29 g / L NaCl; The fermentation culture conditions are as follows: initial pH of 5.0-9.0, shaking speed of 120-240 r / min, inoculation amount of 1%-9%, and temperature of 25-37°C.

[0009] As a further optimization of the high-density fermentation process, the fermentation culture conditions are: initial pH 7.0, shaking speed 180 r / min, inoculation amount 3%, and temperature 34°C.

[0010] In a third aspect, the present invention provides a microbial growth-promoting agent comprising the above-mentioned Sinorhizobium alfalfa H9 and / or its fermentation product.

[0011] In a fourth aspect, the present invention provides the use of the above-mentioned Sinorhizobium medica H9 or microbial growth-promoting agent in promoting plant growth or improving soil performance.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] (1) The Chinese rhizobium meliloti H9 screened out by the present invention has the ability to dissolve insoluble phosphates. After 6 days of cultivation, the soluble phosphorus content in the fermentation liquid can reach 71.17 mg·L -1 .

[0014] (2) The Sinorhizobium meliloti H9 selected by the present invention has strong stress resistance and exhibits strong tolerance to adverse environmental factors such as high salt, acid and alkali, and high temperature. It can tolerate 12% NaCl salt ion concentration and can grow in the pH range of 3 to 12 and the temperature range of 4 to 45°C.

[0015] (3) Since different strains of S. meliloti have different requirements for fermentation medium and fermentation conditions, and there are also large differences in fermentation efficiency, high-density fermentation optimization of S. meliloti H9 was carried out to obtain the optimal fermentation conditions of S. meliloti H9.

[0016] (4) The present invention provides high-density fermentation conditions, which can increase the bacterial density in the fermentation tank and increase the cellular level of the product, thereby correspondingly reducing the volume of the bioreactor, increasing the production capacity of the equipment per unit volume, reducing the separation cost of the biomass, shortening the growth cycle, thereby reducing costs and improving production efficiency.

[0017] Strain preservation description: Sinorhizobium meliloti H9, classified and named Sinorhizobium meliloti, was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on September 9, 2024, with the preservation number CGMCC No.31900. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a comparison chart of the phosphate solubilization ability results of strains H1-H39;

[0019] Figure 2 This is a comparison chart of the phosphate solubilization ability of strain H9 and phosphate solubilizing bacteria Klebsiella variicola PD12;

[0020] Figure 3 is the colony morphology of strain H9;

[0021] Figure 4 is a phylogenetic tree diagram;

[0022] Figure 5 is the growth curve of strain H9;

[0023] Figure 6 This is the result graph showing the effects of initial pH, shaker speed, inoculation volume, liquid volume and temperature on strain growth;

[0024] Figure 7 This is the result diagram of the effect of culture medium components on strain growth;

[0025] Figure 8 are the response surface and contour plot results of the model;

[0026] Fig. 9This is a graph showing the stress resistance of Sinorhizobium lucerne H9. DETAILED DESCRIPTION

[0027] The present invention provides a Sinorhizobium meliloti H9, which is isolated from farmland soil in Changshui Town, Luoning County, Henan Province, China. The present invention measures the phosphorus solubility and stress resistance of Sinorhizobium meliloti H9 and optimizes the fermentation process, and explores the influence of different culture conditions on the biomass of strain H9. The purpose is to obtain the optimal culture process of Sinorhizobium meliloti H9, so as to provide a theoretical basis and experimental basis for further preparing PSB bacterial agents, improving soil performance and increasing crop yields.

[0028] The present invention screens out a strain H9 from farmland soil in Changshui Town, Luoning County, Henan Province, China. The strain forms raised opaque colonies on LB solid culture medium plates, which are round, have neat edges, smooth and moist surfaces, and are yellow in color. The strain isolated by the present invention is sequenced with 16S rDNA sequences, and the sequenced 16S rDNA gene sequences are input into the NCBI website for BLAST analysis. A phylogenetic tree is constructed by MEGA 11.0, and the results show that the H9 strain is homologous to Sinorhizobium meliloti, and H9 belongs to Sinorhizobium meliloti in combination with morphology. The phosphate solubilization ability and stress resistance of H9 are measured, and the results show that the H9 strain is a strain with efficient phosphate solubilization ability and strong stress resistance. The strain is preserved with the preservation number of CGMCC No.31900.

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0030] Example 1: Isolation and purification of strains

[0031] (1) Sample collection: Soil samples were collected from farmland in Changshui Town, Luoning County, Luoyang City, Henan Province, China on March 8, 2024. The average annual temperature in Changshui Town is between 12°C and 14°C, and the annual precipitation is 600-800 mm, mainly concentrated from July to September. Soil samples were collected from the surface layer of 0-5 cm of the farmland profile using a sterilized soil drill. After removing the debris from the surface of the soil, the collected soil samples were immediately placed in sterile bags and sealed, marked, and transported to the laboratory on ice, and stored at 4°C until further analysis.

[0032] (2) Initial screening of phosphate-dissolving bacteria: Weigh 1 g of soil sample using an electronic balance, place it in a sterilized conical flask, and add 9 mL of sterile water. Place it in a shaker at 28°C and 180 r / min for 30 min. After the shaking is completed, let it stand for 30 min. Perform a 10-minute test on a clean bench. -1 , 10-2 , 10 -3 200 μL of gradient dilution was taken respectively and evenly spread on the modified PKO solid medium. The plate was inverted and placed in a constant temperature incubator. It was cultured at 30°C for 6 days. Observe whether a transparent circle appears around the colony. Use an inoculation loop to pick up a single colony with a transparent circle, and repeatedly streak it on the modified PKO solid medium plate for purification until a pure strain is obtained. Store it for later use. The modified PKO solid medium consists of 10 g glucose, 0.5 g (NH 4 ) 2 SO 4 , 0.3 g KCl, 0.3 g MgSO 4 7H 2 O, 0.003gFeSO 4 7H 2 O, 0.003 g MnSO 4 ·4H 2 O, 0.3g NaCl and 15g agar. A total of 20 strains H1-H39 with obvious phosphate solubilization zone were screened out.

[0033] Example 2: Determination of Phosphate Dissolving Capacity

[0034] To evaluate the phosphorus solubilizing ability of the strain, the molybdenum antimony colorimetric method was used to determine the effective phosphorus content in the supernatant. The obtained pure strain H1-H39 was inoculated into LB liquid medium (10 g tryptone, 5 g yeast powder and 10 g NaCl were dissolved in 1000 mL distilled water, pH was about 7.4), and cultured in a shaking incubator at 28 ° C and 180 r / min for 15 h. The bacterial solution concentration was adjusted to OD 600 = about 1, and the seed solution is prepared. The obtained seed solution is inoculated into the modified PKO liquid culture medium at an inoculation rate of 1%, and the culture medium without bacterial solution is used as the control, and cultured at 28°C and 180r / min for 6 days. Shake the culture solution in the shake flask evenly, use a pipette to accurately pipette 4mL of culture solution into a 5mL centrifuge tube, and centrifuge at 10000r / min for 10min. Pipette 1mL of supernatant into a test tube, add 1mL of molybdenum antimony anti-color developer to each tube, shake well, add distilled water to make up to 10mL, use a vortex shaker to mix it thoroughly, and measure the OD after standing at room temperature (26°C) for 30min. 710 Then, the measured absorbance was substituted into the phosphorus standard curve equation y = 0.6562x - 0.0094 (R 2 =0.9993) to obtain the amount of dissolved phosphorus. The result is as follows Figure 1 As shown in Figure 2, the effective phosphorus content in the culture medium is between 0.14 and 71.17 mg·L -1 Among them, H9 has the highest effective phosphorus content (71.17 mg·L -1Compared with the published phosphate-solubilizing bacteria Klebsiella variicola PD12, the H9 strain in this study has excellent phosphate-solubilizing ability ( Figure 2 ). Therefore, H9 was selected as the test strain for subsequent studies.

[0035] Example 3: Identification of strains

[0036] (1) Morphological identification of strains

[0037] H9 was streaked on LB solid medium and cultured at 28°C for 2 days. The biological characteristics of H9 were recorded and the colonies were photographed ( Figure 3 ). It can be seen that the strain forms raised opaque colonies that are round, have neat edges, smooth and moist surfaces, and are yellow in color. Based on the colony morphology, H9 was preliminarily judged to be a bacterium.

[0038] (2) Molecular biological identification of strains

[0039] The bacterial universal primers (27F: 5′-AGAGTTTGATCCTGGCTCAG 3′ and 1492R: 5′-TACGGCTACCTTGTTACGACTT-3′) were used for colony PCR of H9 strain, and the PCR amplification products were detected by 1% agarose gel electrophoresis. The PCR products with obvious bands were sent to Shanghai Biotechnology Co., Ltd. for sequencing. The sequencing results were logged on to the NCBI (National Center for Biotechnology Information) website for BLAST analysis, and ClustalW sequence alignment was performed using MEGA 11.0 software. The phylogenetic tree was constructed using the Neighbor-Joining method. Figure 4 As shown, the H9 strain is homologous to Sinorhizobium meliloti, and combined with morphology, it is judged that H9 belongs to Sinorhizobium meliloti.

[0040] Example 4: Optimization of high-density fermentation of Sinorhizobium meliloti H9

[0041] (1) Determination of strain growth curve

[0042] The activated Sinorhizobium meliloti H9 bacterial solution was inoculated with 3% of the inoculum into NA liquid culture medium (3g beef extract, 10g peptone and 5g NaCl dissolved in 1000mL distilled water, pH controlled to 7.0), and cultured on a shaking table at 28°C and 180r / min for 28h, with sampling every 2h. The blank NA liquid culture medium was used as a control for the test solution, and the OD of the bacterial solution was measured using a UV-visible spectrophotometer. 600 With time as the horizontal axis, OD 600The strain growth curve with the value as the vertical axis is shown in Figure 5 0-2h is the growth lag phase, 2-10h is the logarithmic growth phase, 10-28h is the stable growth phase, and the growth of Sinorhizobium alfalfa H9 tends to be stable at 16h, and the optimal subsequent culture time can be obtained as 16h.

[0043] (2) Optimization of culture conditions

[0044] To explore the effect of culture conditions on strain growth, we mainly considered the effects of different initial pH (5.0, 6.0, 7.0, 8.0, 9.0), shaker speed (120, 140, 180, 220, 240 r / min), inoculation volume (1%, 3%, 5%, 7%, 9%), liquid volume (20, 40, 60, 80, 100 mL) and temperature (25, 28, 31, 34, 37 ° C) on strain growth. Except for the research factors, the other conditions were the same as the basic culture medium and basic fermentation conditions. After shaking culture for 16 hours, the fermentation broth OD was measured. 600 The value of .

[0045] like Figure 6 As shown in the figure, when the initial pH is between 5 and 7, as the initial pH value increases, the OD 600 The value also increased from 0.98±0.01 to 1.14±0.02. When the initial pH was 7, the OD 600 The value reaches the maximum. As the shaking speed increases, the bacterial solution OD 600 The value increased, indicating that strain H9 was aerobic during growth, and the appropriate speed was selected for cultivation in the next culture. 600 The value was 0.85±0.01, which was consistent with the OD of the bacterial solution with inoculation volume of 5%, 7%, and 9%. 600 The maximum difference in value is 0.17. Considering the subsequent factory design, 3% inoculation volume is selected as the best to save costs. When the liquid volume is 20mL, the bacterial growth is the best. The larger the liquid volume, the worse the strain growth. Bacterial liquid OD 600 The value increases first and then decreases with the increase of temperature. When the temperature is 37℃, the OD 600 The value is low, and the bacteria grow slowly. Under the temperature condition of 25-34℃, the OD 600 The overall value is high, indicating that the bacteria grow well under this condition. When the temperature is 34℃, the OD 600 value reaches the maximum value.

[0046] (3) Optimization of culture medium components

[0047] To explore the effects of culture medium components on strain growth, the effects of different carbon sources, nitrogen sources and inorganic salts on strain growth at different concentrations were mainly investigated. Sucrose, mannitol, lactose, glucose and maltose were used to replace the carbon source (beef extract) in NA liquid medium. The concentrations of each carbon source were 1% (w / v), 5% (w / v) and 9% (w / v). When studying nitrogen sources, potassium nitrate, ammonium chloride, ammonium sulfate, yeast powder and peptone were selected as nitrogen sources based on NA liquid medium without peptone. The concentrations of each nitrogen source were 0.5% (w / v), 1% (w / v) and 1.5% (w / v). Magnesium chloride, ferric chloride, sodium chloride, zinc chloride and manganese chloride were used to replace inorganic salts in NA liquid medium. The concentrations of each inorganic salt were 0.1% (w / v), 0.55% (w / v) and 1% (w / v). The fermentation experiment used a 50mL conical flask with a liquid volume of 20mL and adjusted the initial pH to 7.0. The H9 seed liquid was inoculated into the fermentation medium at a rate of 3%, and the fermentation liquid was shaken and cultured at 28°C and 180 r / min for 16 h, and then the OD of the fermentation liquid was measured. 600 The value of .

[0048] Depend on Figure 7 It can be seen that the biomass of the fermentation broth of strain H9 was the highest when 5% lactose was used as the carbon source. 600 The value was 1.10±0.02. The OD of the fermentation liquid after adding organic nitrogen source 600 The value was significantly higher than that of the fermentation broth with inorganic nitrogen source added. Among them, strain H9 had the strongest ability to utilize yeast powder, followed by peptone. When 1.0% yeast powder was used as the nitrogen source, the OD of strain H9 was 600 When the inorganic salt in the culture medium is sodium chloride or magnesium chloride, the bacterial solution OD 600 The value was significantly higher than that of the other three inorganic salts. When 0.55% sodium chloride was used as the inorganic salt, the OD 600 The value (1.32±0.01) was the highest.

[0049] Example 5: Response surface methodology optimization

[0050] The Box-Behnken response surface model in Design-Expert 13 software was used to optimize the type and concentration of carbon source, type and concentration of nitrogen source, and type and concentration of inorganic salt. According to the results of the single factor experiment, the optimal culture conditions were selected, and the viable count of strain H9 was used as the response value. The lactose concentration (A), yeast powder concentration (B), and sodium chloride concentration (C) were used as the investigation factors. According to the Box-Behnken experimental design principle, Design-Expert 13 was used to design 17 groups of experiments with three factors and three levels, and the viable count of strain H9 under each condition was determined. The Box-Benhnken experimental design factors and levels are shown in Table 1. The Box-Benhnken experimental design and results are shown in Table 2.

[0051] Table 1: Box-Benhnken test design factors and levels

[0052] Table 2: Box-Benhnken test design and results

[0053] According to the results of the Box-Behnken test, the test data were fitted with multiple regression, and a three-variable second-order polynomial regression model of viable count (Y) on lactose concentration (A), yeast powder concentration (B) and sodium chloride concentration (C) was established. In order to evaluate the interaction between the factors, case statistical diagnosis was used to check the fit of the statistical model. The F test was used to verify the validity of the model, and the R 2 The coefficients were used to evaluate the significance of the model fit. Finally, the composition parameters of the culture medium were statistically optimized using analysis of variance (ANOVA), and the optimal conditions were verified. The statistical significance of the experiment was based on a p value < 0.05 (only when the p value was less than 0.05, the results were considered statistically significant).

[0054] According to the Box-Behnken central composite experimental design principle, the obtained ternary quadratic fitting regression equation is: Y = -0.417866 + 0.0663568 × A + 0.076489 × B - 0.0055054 × C + 0.00002 × AB + 0.000505 × AC - 0.000255 × BC - 0.000691 × A 2 -0.003498×B 2 -0.002465×C 2 , where Y represents the viable count of strain H9, and A, B, and C correspond to colostrum concentration, yeast powder concentration, and sodium chloride concentration, respectively.

[0055] The regression equation was subjected to variance analysis, and the results are shown in Table 3. The F-value of the model is 34.06 (p<0.0001) and the F-value of the lack-of-fit term is 2.23 (p=0.2267>0.05), so the fit of the model reaches a significant level. The F value indicates the degree of influence of several factors in the fitting model on the response value. The larger the F value, the greater the influence of the factor on the response value. According to the F value, the influence of the three factors on the viable count of strain H9 is ranked, and the order of influence is: C>B>A, that is, sodium chloride concentration>yeast powder concentration>lactose concentration. P-value less than 0.05 indicates that the model term is significant. In this case, the linear term (A, B, C), the quadratic term (A) of lactose concentration (A), yeast powder concentration (B) and sodium chloride concentration (C) 2 , B2 , C 2 ) had a very significant effect on the viable count of strain H9 (p<0.01). Among the interaction terms, the interaction between lactose concentration and sodium chloride concentration (AC) had a significant effect on the viable count of strain H9 (p<0.05). The interaction between lactose concentration and yeast powder concentration (AB) had no significant effect on the viable count of strain H9 (p>0.05). The interaction between yeast powder concentration and sodium chloride concentration (BC) had no significant effect on the viable count of strain H9 (p>0.05). R 2 =0.9777, indicating that the probability that the regression model can explain the number of live bacteria of strain H9 is 97.77%, indicating that the model has high credibility and can be used for simulation prediction of the number of live bacteria in fermentation test of strain H9.

[0056] Table 3: Analysis of variance

[0057] The response surface and contour plot results of the model are shown in Figure 8 As shown in the figure, each figure shows the effect of the interaction of any two variables among lactose concentration, yeast powder concentration, and sodium chloride concentration on the viable count of strain H9. From the response surface analysis diagram, it can be seen intuitively that the strength of the interaction between the two variables is mainly reflected by the shape of the response surface curve. The steeper the slope of the response surface curve, the closer the contour line graph is to the ellipse, indicating that the interaction between the two variables is more significant; otherwise, it means that the interaction is not significant. The results show that the response surface curvature of lactose concentration and sodium chloride concentration is the largest, and the two have the most significant effect on the viable count of strain H9.

[0058] Figure 8 a, b are the effects of the interaction between lactose concentration and yeast powder concentration on the viable count of strain H9 when the sodium chloride concentration is at the central level, i.e., 5.5 g / L. As can be seen from the figure, the viable count first increases and then decreases with the increase of lactose concentration and yeast powder concentration. The contour shape is the least close to an ellipse, and the interaction between the two is not significant.

[0059] Figure 8 c, d are the effects of the interaction between lactose concentration and sodium chloride concentration on the viable count of strain H9 when the yeast powder concentration is at the central level, i.e., 10 g / L. From this figure, it can be seen that the slope of the response surface of the interaction between lactose concentration and sodium chloride concentration is steep, and the viable count first increases and then decreases with the increase of lactose concentration and sodium chloride concentration. And its contour shape is closest to an ellipse, and the interaction between the two is the most significant.

[0060] Figure 8e,f are the effects of the interaction between yeast powder concentration and sodium chloride concentration on the viable count of strain H9 when the lactose concentration is at the central level, i.e., 50 g / L. As can be seen from the figure, the contour map is not close to an ellipse, and the interaction between the two is not significant. Although the effect is not significant, when one variable is fixed, the viable count first increases and then decreases with the increase of the other variable, but the change is not significant.

[0061] The experimental data were processed and the model response value was predicted by Design-Expert 13 software. At the same time, the response surface plot and contour line analysis showed that the optimal culture process for the fermentation test of strain H9 was: lactose concentration of 48g / L, yeast powder concentration of 11g / L, and sodium chloride concentration of 4g / L. Under this condition, the live bacterial count of strain H9 was predicted to reach 14.971×10 9 CFU / mL. Combining the results of single factor experiment and response surface optimization experiment, the optimal culture conditions for strain H9 fermentation were determined as follows: lactose 48.32g / L, yeast powder 11.32g / L, NaCl 3.29g / L, initial pH controlled at 7.0, liquid volume 20mL, inoculation volume 3%, shaker set at 34℃, 180r / min. Under these conditions, the fermentation experiment of strain H9 was carried out, and three groups of parallel experiments were set up. The average value of the viable count of strain H9 under the optimal culture process was (1.490±0.05)×10 10 CFU / mL, compared with the predicted value of 1.497×10 10 CFU / mL is close. Therefore, it shows that the experimental results fit the model well, indicating that the optimization of the viable count of strain H9 using response surface methodology is accurate and reliable, and can truly reflect the impact of each variable on the viable count of strain H9.

[0062] Example 6: Determination of stress resistance of Sinorhizobium meliloti H9

[0063] The stress resistance of Sinorhizobium alfalfa H9 was tested in terms of salt resistance, acid resistance, alkali resistance and growth temperature range. Based on YMA medium, the H9 strain was inoculated with 2% inoculation amount by adjusting different salt concentrations and pH values. After culturing at 28°C and 180rpm / min for 2-5 days, the strain was inoculated on a YMA plate and the growth of H9 was observed every 3 hours. The heat shock treatment of the strain was carried out by the coating method.

[0064] YMA solid medium (g / L): 10 g mannitol, 0.1 g NaCl, 0.2 g MgSO 4 7H 2 O, 1.0g yeast powder, 0.5g K 2 HPO 4 , 16 g agar powder, 1000 mL distilled water, pH 7.0.

[0065] (1) Salt tolerance determination: YMA culture medium containing NaCl was prepared, wherein the mass volume fraction of NaCl was 5%, 6%, 7%, 8%, 9%, 10%, 11%, and 12%.

[0066] (2) Acid resistance determination: The initial pH value of the YMA culture medium was adjusted with HCl to 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0, respectively.

[0067] (3) Alkali resistance determination: The initial pH value of the YMA culture medium was adjusted with NaOH to 8.0, 9.0, 10.0, 11.0, 12.0, and 13.0, respectively.

[0068] (4) Determination of growth temperature range: H9 seed solution was inoculated on YMA plate culture medium by the spot grafting method and cultured in a constant temperature incubator at 4°C, 10°C, 15°C, 25°C, 28°C, 35°C, 40°C, and 45°C for 3 days to observe and record the colony growth.

[0069] (5) Heat shock treatment: The H9 seed solution was heat shocked at 60°C for 10 min, 12 min, and 15 min; and at 62°C for 10 min, 12 min, and 15 min, respectively. Then, the solution was spread on YMA solid medium and cultured at 28°C for 3 days. The growth of the colonies was observed and recorded.

[0070] Test results ( Fig. 9 ) showed that Sinorhizobium alfalfa H9 had strong stress resistance. It had strong salt tolerance and could grow in a culture medium containing 12% NaCl. It had strong acid-base tolerance and could grow in a culture medium with a pH of 3 to 13. The growth temperature range of strain H9 was wide, and it could grow in a temperature range of 4 to 45°C. Moreover, the strain could still survive after heat shock treatment at 62°C for 15 minutes, indicating that strain H9 could tolerate high temperature for a short period of time.

[0071] It should be noted that the above-described embodiments should be understood as illustrative rather than limiting the scope of protection of the present invention, and the scope of protection of the present invention shall be subject to the claims. For those skilled in the art, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention without departing from the essence and scope of the present invention.

Claims

1. A Sinorhizobium meliloti H9, classification and naming Sinorhizobium meliloti , and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 9, 2024, with the deposit number CGMCC No.31900.

2. The high-density fermentation process of Sinorhizobium meliloti H9 according to claim 1, characterized in that: Inoculating Sinorhizobium meliloti H9 into a fermentation medium for fermentation culture; The fermentation medium contains: 48.32 g / L lactose, 11.32 g / L yeast powder, and 3.29 g / L NaCl; The fermentation culture conditions are: initial pH 5.0-9.0, shaking speed 120-240 r / min, inoculation amount 1%-9%, and temperature 25-37°C.

3. The high-density fermentation process according to claim 2, characterized in that: The fermentation culture conditions are: initial pH 7.0, shaking speed 180 r / min, inoculation amount 3%, and temperature 34°C.

4. A microbial growth-promoting agent, comprising the Sinorhizobium alfalfa H9 described in claim 1 and / or its fermentation product.

5. Use of the Sinorhizobium meliloti H9 according to claim 1 or the microbial growth-promoting agent according to claim 4 in promoting plant growth or improving soil performance.

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