Culture medium and preparation method for screening nitrogen-fixing and aluminum-resistant nitrogen-fixing bacteria.

Abstract

This invention discloses a culture medium for simultaneously screening nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria, and its preparation method. The medium uses aluminum chloride, spinach juice, and rapeseed juice, along with a low pH value, to simulate an aluminum-tolerant environment. Spinach and rapeseed juices provide abundant aluminum, micronutrients, physiologically active substances, and growth hormones, ensuring that this aluminum-tolerant environment does not have a toxic effect on the nitrogen-fixing bacteria. This allows for the simultaneous screening of nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria, with the highest total aluminum tolerance of the screened aluminum-tolerant bacteria reaching 5000–5500 μg / g. Adding appropriate amounts of activated carbon powder, bagasse powder, and sugarcane leaf powder effectively improves the aeration of the agar medium and effectively adsorbs the metabolic waste excreted by both nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria, preventing the metabolic waste from having a toxic effect on these bacteria. This facilitates the simultaneous screening of nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria and significantly improves the yield of these bacteria.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, specifically to a culture medium for simultaneously screening nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria and its preparation method. Background Technology

[0002] Sugarcane, also known as sweet sugarcane, stalk sugarcane, sugar stalk, dried sugarcane, stalk sugarcane, bamboo sugarcane, and intestinal herb, belongs to the Poaceae family, Sorghum tribe, and Canesmus genus. It is a tall, vigorous, monocotyledonous annual or perennial herbaceous plant native to tropical and subtropical regions. The sugarcane stalk is solid, growing regularly like bamboo, with nodes, each node containing a bud. Sugarcane grows in dense clumps, with beautiful, sword-shaped leaves similar to corn leaves. The leaf sheaths wrap around the stalk, protecting the buds. The stalks of sugarcane are rich in sweet juice, which is the raw material for making sugar and can also be used to extract ethanol as an energy substitute.

[0003] Soil aluminum toxicity is a serious agricultural production problem that is prevalent worldwide, affecting crop production on 40% of the world's arable land. Acidic soils pose certain obstacles to plant growth and development, affecting not only plant yield and quality but also all life activities of plants. The main manifestations are: (1) H + High concentration produces H + (1) High concentration of active aluminum leads to aluminum toxicity; (2) High concentration of active manganese leads to manganese toxicity, thus limiting plant growth; (3) Low cation availability leads to magnesium, calcium, and potassium deficiency; (4) Inhibited cation absorption leads to magnesium, calcium, and potassium deficiency; (5) Reduced solubility of phosphorus and molybdenum leads to phosphorus and molybdenum deficiency; (6) Inhibited root growth; (7) Increased leaching leads to nutrient loss. These obstacles seriously affect plant growth and development. Research on aluminum toxicity in plants has always been a hot topic.

[0004] To mitigate aluminum toxicity to sugarcane, nitrogen-fixing bacteria with nitrogen-fixing and aluminum-tolerant characteristics can be screened from the soil. By utilizing the nitrogen-fixing activity of these bacteria, as well as their absorption and adsorption of active aluminum in acidic soil and the chelation of aluminum ions by their secretions, the toxicity of aluminum to sugarcane can be effectively alleviated. This measure offers long-lasting and low-cost benefits. Currently, there is no culture medium that can simultaneously screen for nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a culture medium and its preparation method that can simultaneously screen nitrogen-fixing and aluminum-resistant nitrogen-fixing bacteria, is low in cost, and has a simple preparation method.

[0006] To achieve the above objectives, the technical solution adopted by this invention is as follows: a culture medium for simultaneously screening nitrogen-fixing and aluminum-resistant nitrogen-fixing bacteria, wherein the culture medium comprises: 15 g / L agar, 8 g / L glucose, 3 g / L sucrose, 4 g / L peptone, 1 g / L soluble starch, 0.5 g / L urea, 1.2 g / L sodium chloride, 0.4 g / L magnesium sulfate, and 1.3 g / L dihydrogen phosphate. Potassium, 0.2 g / L calcium chloride, 0.1 g / L aluminum chloride, 0.1 g / L manganese chloride, 0.1 g / L sodium molybdate, 1.2–1.5 g / L activated carbon powder, 0.4–0.7 g / L sugarcane bagasse powder, 0.3–0.5 g / L sugarcane leaf powder, 35–40 ml / L spinach juice, 40–45 ml / L rapeseed juice, with the pH controlled at 5.5–6.5, and the remainder being water;

[0007] The pH value of the culture medium is controlled using citric acid and sodium citrate.

[0008] The spinach juice contains 80-100 mg / kg of aluminum, and the rapeseed juice contains 60-80 mg / kg of aluminum.

[0009] Furthermore, a culture medium for simultaneously screening nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria was developed. The culture medium consisted of: 15 g / L agar, 8 g / L glucose, 3 g / L sucrose, 4 g / L peptone, 1 g / L soluble starch, 0.5 g / L urea, 1.2 g / L sodium chloride, 0.4 g / L magnesium sulfate, 1.3 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, 0.1 g / L aluminum chloride, 0.1 g / L manganese chloride, 0.1 g / L sodium molybdate, 1.3–1.5 g / L activated carbon powder, 0.5–0.7 g / L bagasse powder, 0.4–0.5 g / L sugarcane leaf powder, 37–40 ml / L spinach juice, and 42–45 ml / L rapeseed juice. The pH was controlled at 5.5–6.5, with the remainder being water.

[0010] The pH value of the culture medium is controlled using citric acid and sodium citrate.

[0011] The spinach juice contains 80-100 mg / kg of aluminum, and the rapeseed juice contains 60-80 mg / kg of aluminum.

[0012] Furthermore, a culture medium for simultaneously screening nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria was developed. The culture medium consisted of: 15 g / L agar, 8 g / L glucose, 3 g / L sucrose, 4 g / L peptone, 1 g / L soluble starch, 0.5 g / L urea, 1.2 g / L sodium chloride, 0.4 g / L magnesium sulfate, 1.3 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, 0.1 g / L aluminum chloride, 0.1 g / L manganese chloride, 0.1 g / L sodium molybdate, 1.4 g / L activated carbon powder, 0.6 g / L bagasse powder, 0.45 g / L sugarcane leaf powder, 38 ml / L spinach juice, and 43 ml / L rapeseed juice. The pH was controlled at 5.5–5.8, with the remainder being water.

[0013] The pH value of the culture medium is controlled using citric acid and sodium citrate.

[0014] The spinach juice contains 80-100 mg / kg of aluminum, and the rapeseed juice contains 60-80 mg / kg of aluminum.

[0015] Furthermore, the spinach juice is freshly squeezed spinach juice, and the rapeseed juice is freshly squeezed rapeseed juice.

[0016] Furthermore, the culture medium also includes 0.4–0.6 g / L of quinoa powder.

[0017] Furthermore, the culture medium also includes 0.5 g / L of quinoa powder.

[0018] This invention also provides a method for preparing a culture medium for simultaneously screening nitrogen-fixing and aluminum-resistant nitrogen-fixing bacteria, comprising the following steps:

[0019] (1) Preparation of spinach juice and rapeseed juice:

[0020] 1.1) Select fresh spinach, cut off the roots, wash it clean, press it into juice, filter it, and obtain spinach juice;

[0021] 1.2) Select fresh rapeseed, wash it clean, press it into juice, filter it to obtain rapeseed juice;

[0022] (2) Weighing: Weigh each raw material that makes up the culture medium according to the ratio;

[0023] (3) Mixing and preparation: Mix all the raw materials that make up the culture medium evenly, and use citric acid and sodium citrate to control the pH value at 5.5 to 6.5 to obtain a mixed solution;

[0024] (4) Sterilization: The mixture is sterilized to obtain the culture medium.

[0025] Further, in step (1), spinach juice and rapeseed juice are prepared:

[0026] 1.1) Select fresh spinach with an aluminum content of about 120 mg / kg, cut off the roots, wash it clean, press it into juice, filter it to obtain spinach juice with an aluminum content of 80-100 mg / kg.

[0027] 1.2) Select fresh rapeseed with an aluminum content of about 100 mg / kg, wash it clean, press it into juice, filter it to obtain rapeseed juice with an aluminum content of 60-80 mg / kg.

[0028] Furthermore, the filter membrane used in step (1) to prepare spinach juice and rapeseed juice is a 0.22μm sterile filter membrane.

[0029] Further, in step (4), the mixture is sterilized for 30 minutes at a temperature of 115°C and a pressure of 0.5 MPa to obtain the culture medium.

[0030] This invention discloses a culture medium for simultaneously screening nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria, and its preparation method. The medium uses aluminum chloride, spinach juice, and rapeseed juice, along with a low pH value, to simulate an aluminum-tolerant environment. Spinach and rapeseed juices provide abundant aluminum, micronutrients, physiologically active substances, and growth hormones, ensuring that this aluminum-tolerant environment does not have a toxic effect on the nitrogen-fixing bacteria. This allows for the simultaneous screening of nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria, with the highest total aluminum tolerance of the screened aluminum-tolerant bacteria reaching 5000–5500 μg / g. Adding appropriate amounts of activated carbon powder, bagasse powder, and sugarcane leaf powder effectively improves the aeration of the agar medium and effectively adsorbs the metabolic waste excreted by both nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria, preventing the metabolic waste from having a toxic effect on these bacteria. This facilitates the simultaneous screening of nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria and significantly improves the yield of these bacteria.

[0031] This invention discloses a culture medium for screening nitrogen-fixing bacteria with both nitrogen-fixing and aluminum-resistant properties, and a method for preparing the same. The culture medium also contains an appropriate amount of quinoa powder, which can enhance the nitrogen-fixing ability of the nitrogen-fixing bacteria. Detailed Implementation

[0032] The following embodiments can help those skilled in the art to more fully understand the present invention, but should not be construed as limiting the present invention in any way.

[0033] Example 1

[0034] Simultaneously, a culture medium for screening nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria was developed. The culture medium consisted of: 15 g / L agar, 8 g / L glucose, 3 g / L sucrose, 4 g / L peptone, 1 g / L soluble starch, 0.5 g / L urea, 1.2 g / L sodium chloride, 0.4 g / L magnesium sulfate, 1.3 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, 0.1 g / L aluminum chloride, 0.1 g / L manganese chloride, 0.1 g / L sodium molybdate, 1.2 g / L activated carbon powder, 0.4 g / L bagasse powder, 0.3 g / L sugarcane leaf powder, 35 ml / L spinach juice, 40 ml / L rapeseed juice, and 0.4 g / L quinoa powder. The pH was controlled at 5.5, with the remainder being water. The spinach juice and rapeseed juice were freshly squeezed.

[0035] The pH value of the culture medium is controlled using citric acid and sodium citrate.

[0036] The spinach juice contains 80-100 mg / kg of aluminum, and the rapeseed juice contains 60-80 mg / kg of aluminum.

[0037] A method for preparing a culture medium for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-resistant properties includes the following steps:

[0038] (1) Preparation of spinach juice and rapeseed juice:

[0039] 1.1) Select fresh spinach with an aluminum content of about 120 mg / kg, cut off the roots, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain spinach juice with an aluminum content of 80-100 mg / kg.

[0040] 1.2) Select fresh rapeseed with an aluminum content of about 100 mg / kg, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain rapeseed juice with an aluminum content of 60-80 mg / kg.

[0041] (2) Weighing: Weigh each raw material that makes up the culture medium according to the ratio;

[0042] (3) Mixing and preparation: Mix all the raw materials that make up the culture medium evenly, and use citric acid and sodium citrate to control the pH value at 5.5 to obtain a mixed solution;

[0043] (4) Sterilization: The mixture is sterilized at a temperature of 115℃ and a pressure of 0.5Mpa for 30 minutes to obtain the culture medium.

[0044] The method for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-tolerant properties using the culture medium includes the following steps:

[0045] (1) Collect sugarcane rhizosphere soil and prepare sugarcane rhizosphere soil suspension;

[0046] (2) The sugarcane rhizosphere soil suspension was placed in the culture medium and cultured to form colonies, and the strain was obtained;

[0047] (3) The strain was purified and identified, showing that the culture medium could simultaneously screen for nitrogen-fixing bacteria with nitrogen-fixing and acid-aluminum-resistant characteristics.

[0048] Example 2

[0049] Simultaneously, a culture medium for screening nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria was developed. The culture medium consisted of: 15 g / L agar, 8 g / L glucose, 3 g / L sucrose, 4 g / L peptone, 1 g / L soluble starch, 0.5 g / L urea, 1.2 g / L sodium chloride, 0.4 g / L magnesium sulfate, 1.3 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, 0.1 g / L aluminum chloride, 0.1 g / L manganese chloride, 0.1 g / L sodium molybdate, 1.5 g / L activated carbon powder, 0.7 g / L bagasse powder, 0.5 g / L sugarcane leaf powder, 40 ml / L spinach juice, 45 ml / L rapeseed juice, and 0.6 g / L quinoa powder. The pH was controlled at 6.5, with the remainder being water. The spinach juice and rapeseed juice were freshly squeezed.

[0050] The pH value of the culture medium is controlled using citric acid and sodium citrate.

[0051] The spinach juice contains 80-100 mg / kg of aluminum, and the rapeseed juice contains 60-80 mg / kg of aluminum.

[0052] A method for preparing a culture medium for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-resistant properties includes the following steps:

[0053] (1) Preparation of spinach juice and rapeseed juice:

[0054] 1.1) Select fresh spinach with an aluminum content of about 120 mg / kg, cut off the roots, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain spinach juice with an aluminum content of 80-100 mg / kg.

[0055] 1.2) Select fresh rapeseed with an aluminum content of about 100 mg / kg, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain rapeseed juice with an aluminum content of 60-80 mg / kg.

[0056] (2) Weighing: Weigh each raw material that makes up the culture medium according to the ratio;

[0057] (3) Mixing and preparation: Mix all the raw materials that make up the culture medium evenly, and use citric acid and sodium citrate to control the pH value at 6.5 to obtain a mixed solution;

[0058] (4) Sterilization: The mixture is sterilized at a temperature of 115℃ and a pressure of 0.5Mpa for 30 minutes to obtain the culture medium.

[0059] The method for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-tolerant properties using the culture medium includes the following steps:

[0060] (1) Collect sugarcane rhizosphere soil and prepare sugarcane rhizosphere soil suspension;

[0061] (2) The sugarcane rhizosphere soil suspension was placed in the culture medium and cultured to form colonies, and the strain was obtained;

[0062] (3) The strain was purified and identified, showing that the culture medium could simultaneously screen for nitrogen-fixing bacteria with nitrogen-fixing and acid-aluminum-resistant characteristics.

[0063] Example 3

[0064] Simultaneously, a culture medium was used to screen nitrogen-fixing and aluminum-resistant nitrogen-fixing bacteria. The culture medium consisted of: 15 g / L agar, 8 g / L glucose, 3 g / L sucrose, 4 g / L peptone, 1 g / L soluble starch, 0.5 g / L urea, 1.2 g / L sodium chloride, 0.4 g / L magnesium sulfate, 1.3 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, and 0.1 g / L... The mixture consists of aluminum chloride, 0.1 g / L manganese chloride, 0.1 g / L sodium molybdate, 1.3 g / L activated carbon powder, 0.5 g / L bagasse powder, 0.4 g / L sugarcane leaf powder, 37 ml / L spinach juice, 42 ml / L rapeseed juice, and 0.56 g / L quinoa powder, with the pH controlled at 5.8, and the remainder being water; the spinach juice is freshly squeezed spinach juice, and the rapeseed juice is freshly squeezed rapeseed juice.

[0065] The pH value of the culture medium is controlled using citric acid and sodium citrate.

[0066] The spinach juice contains 80-100 mg / kg of aluminum, and the rapeseed juice contains 60-80 mg / kg of aluminum.

[0067] A method for preparing a culture medium for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-resistant properties includes the following steps:

[0068] (1) Preparation of spinach juice and rapeseed juice:

[0069] 1.1) Select fresh spinach with an aluminum content of about 120 mg / kg, cut off the roots, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain spinach juice with an aluminum content of 80-100 mg / kg.

[0070] 1.2) Select fresh rapeseed with an aluminum content of about 100 mg / kg, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain rapeseed juice with an aluminum content of 60-80 mg / kg.

[0071] (2) Weighing: Weigh each raw material that makes up the culture medium according to the ratio;

[0072] (3) Mixing and preparation: Mix all the raw materials that make up the culture medium evenly, and use citric acid and sodium citrate to control the pH value at 5.8 to obtain a mixed solution;

[0073] (4) Sterilization: The mixture is sterilized at a temperature of 115℃ and a pressure of 0.5Mpa for 30 minutes to obtain the culture medium.

[0074] The method for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-tolerant properties using the culture medium includes the following steps:

[0075] (1) Collect sugarcane rhizosphere soil and prepare sugarcane rhizosphere soil suspension;

[0076] (2) The sugarcane rhizosphere soil suspension was placed in the culture medium and cultured to form colonies, and the strain was obtained;

[0077] (3) The strain was purified and identified, showing that the culture medium could simultaneously screen for nitrogen-fixing bacteria with nitrogen-fixing and acid-aluminum-resistant characteristics.

[0078] Example 4

[0079] Simultaneously, a culture medium was used to screen nitrogen-fixing and aluminum-resistant nitrogen-fixing bacteria. The culture medium consisted of: 15 g / L agar, 8 g / L glucose, 3 g / L sucrose, 4 g / L peptone, 1 g / L soluble starch, 0.5 g / L urea, 1.2 g / L sodium chloride, 0.4 g / L magnesium sulfate, 1.3 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, and 0.1 g / L... The mixture consists of aluminum chloride, 0.1 g / L manganese chloride, 0.1 g / L sodium molybdate, 1.4 g / L activated carbon powder, 0.6 g / L bagasse powder, 0.45 g / L sugarcane leaf powder, 38 ml / L spinach juice, 43 ml / L rapeseed juice, and 0.5 g / L quinoa powder, with the pH controlled at 5.8, and the remainder being water; the spinach juice is freshly squeezed spinach juice, and the rapeseed juice is freshly squeezed rapeseed juice.

[0080] The pH value of the culture medium is controlled using citric acid and sodium citrate.

[0081] The spinach juice contains 80-100 mg / kg of aluminum, and the rapeseed juice contains 60-80 mg / kg of aluminum.

[0082] A method for preparing a culture medium for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-resistant properties includes the following steps:

[0083] (1) Preparation of spinach juice and rapeseed juice:

[0084] 1.1) Select fresh spinach with an aluminum content of about 120 mg / kg, cut off the roots, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain spinach juice with an aluminum content of 80-100 mg / kg.

[0085] 1.2) Select fresh rapeseed with an aluminum content of about 100 mg / kg, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain rapeseed juice with an aluminum content of 60-80 mg / kg.

[0086] (2) Weighing: Weigh each raw material that makes up the culture medium according to the ratio;

[0087] (3) Mixing and preparation: Mix all the raw materials that make up the culture medium evenly, and use citric acid and sodium citrate to control the pH value at 5.8 to obtain a mixed solution;

[0088] (4) Sterilization: The mixture is sterilized at a temperature of 115℃ and a pressure of 0.5Mpa for 30 minutes to obtain the culture medium.

[0089] The method for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-tolerant properties using the culture medium includes the following steps:

[0090] (1) Collect sugarcane rhizosphere soil and prepare sugarcane rhizosphere soil suspension;

[0091] (2) The sugarcane rhizosphere soil suspension was placed in the culture medium and cultured to form colonies, and the strain was obtained;

[0092] (3) The strain was purified and identified, showing that the culture medium could simultaneously screen for nitrogen-fixing bacteria with nitrogen-fixing and acid-aluminum-resistant characteristics.

[0093] Example 5

[0094] Simultaneously, a culture medium for screening nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria was developed. The culture medium consisted of: 15 g / L agar, 8 g / L glucose, 3 g / L sucrose, 4 g / L peptone, 1 g / L soluble starch, 0.5 g / L urea, 1.2 g / L sodium chloride, 0.4 g / L magnesium sulfate, 1.3 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, 0.1 g / L aluminum chloride, 0.1 g / L manganese chloride, 0.1 g / L sodium molybdate, 1.4 g / L activated carbon powder, 0.6 g / L bagasse powder, 0.45 g / L sugarcane leaf powder, 38 ml / L spinach juice, and 43 ml / L rapeseed juice, with the pH controlled at 5.8, and the remainder being water. The spinach juice and rapeseed juice were both freshly squeezed.

[0095] The pH value of the culture medium is controlled using citric acid and sodium citrate.

[0096] The spinach juice contains 80-100 mg / kg of aluminum, and the rapeseed juice contains 60-80 mg / kg of aluminum.

[0097] A method for preparing a culture medium for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-resistant properties includes the following steps:

[0098] (1) Preparation of spinach juice and rapeseed juice:

[0099] 1.1) Select fresh spinach with an aluminum content of about 120 mg / kg, cut off the roots, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain spinach juice with an aluminum content of 80-100 mg / kg.

[0100] 1.2) Select fresh rapeseed with an aluminum content of about 100 mg / kg, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain rapeseed juice with an aluminum content of 60-80 mg / kg.

[0101] (2) Weighing: Weigh each raw material that makes up the culture medium according to the ratio;

[0102] (3) Mixing and preparation: Mix all the raw materials that make up the culture medium evenly, and use citric acid and sodium citrate to control the pH value at 5.8 to obtain a mixed solution;

[0103] (4) Sterilization: The mixture is sterilized at a temperature of 115℃ and a pressure of 0.5Mpa for 30 minutes to obtain the culture medium.

[0104] The method for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-tolerant properties using the culture medium includes the following steps:

[0105] (1) Collect sugarcane rhizosphere soil and prepare sugarcane rhizosphere soil suspension;

[0106] (2) The sugarcane rhizosphere soil suspension was placed in the culture medium and cultured to form colonies, and the strain was obtained;

[0107] (3) The strain was purified and identified, showing that the culture medium could simultaneously screen for nitrogen-fixing bacteria with nitrogen-fixing and acid-aluminum-resistant characteristics.

[0108] Comparative Example 1

[0109] Simultaneously, a culture medium for screening nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria was developed. The culture medium consisted of: 15 g / L agar, 8 g / L glucose, 3 g / L sucrose, 4 g / L peptone, 1 g / L soluble starch, 0.5 g / L urea, 1.2 g / L sodium chloride, 0.4 g / L magnesium sulfate, 1.3 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, 0.1 g / L aluminum chloride, 0.1 g / L manganese chloride, 0.1 g / L sodium molybdate, 38 ml / L spinach juice, 43 ml / L rapeseed juice, and 0.5 g / L quinoa flour. The pH was controlled at 5.8, with the remainder being water. The spinach juice and rapeseed juice were both freshly squeezed.

[0110] The pH value of the culture medium is controlled using citric acid and sodium citrate.

[0111] The spinach juice contains 80-100 mg / kg of aluminum, and the rapeseed juice contains 60-80 mg / kg of aluminum.

[0112] A method for preparing a culture medium for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-resistant properties includes the following steps:

[0113] (1) Preparation of spinach juice and rapeseed juice:

[0114] 1.1) Select fresh spinach with an aluminum content of about 120 mg / kg, cut off the roots, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain spinach juice with an aluminum content of 80-100 mg / kg.

[0115] 1.2) Select fresh rapeseed with an aluminum content of about 100 mg / kg, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain rapeseed juice with an aluminum content of 60-80 mg / kg.

[0116] (2) Weighing: Weigh each raw material that makes up the culture medium according to the ratio;

[0117] (3) Mixing and preparation: Mix all the raw materials that make up the culture medium evenly, and use citric acid and sodium citrate to control the pH value at 5.8 to obtain a mixed solution;

[0118] (4) Sterilization: The mixture is sterilized at a temperature of 115℃ and a pressure of 0.5Mpa for 30 minutes to obtain the culture medium.

[0119] The method for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-tolerant properties using the culture medium includes the following steps:

[0120] (1) Collect sugarcane rhizosphere soil and prepare sugarcane rhizosphere soil suspension;

[0121] (2) The sugarcane rhizosphere soil suspension was placed in the culture medium and cultured to form colonies, and the strain was obtained;

[0122] (3) The strain was purified and identified, and it was found that the acid-aluminum-resistant nitrogen-fixing bacteria could not be screened out through the culture medium.

[0123] Comparative Example 2

[0124] Simultaneously, a culture medium for screening nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria was established. The culture medium consisted of: 15 g / L agar, 8 g / L glucose, 3 g / L sucrose, 4 g / L peptone, 1 g / L soluble starch, 0.5 g / L urea, 1.2 g / L sodium chloride, 0.4 g / L magnesium sulfate, 1.3 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, 0.2 g / L aluminum chloride, 0.1 g / L manganese chloride, 0.1 g / L sodium molybdate, 1.4 g / L activated carbon powder, 0.6 g / L bagasse powder, 0.45 g / L sugarcane leaf powder, and 0.5 g / L quinoa powder. The pH was controlled at 5.8, with the remainder being water.

[0125] The pH value of the culture medium is controlled using citric acid and sodium citrate.

[0126] A method for preparing a culture medium for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-resistant properties includes the following steps:

[0127] (1) Weighing: Weigh each raw material that makes up the culture medium according to the ratio;

[0128] (2) Mixing and preparation: Mix all the raw materials that make up the culture medium evenly, and use citric acid and sodium citrate to control the pH value at 5.8 to obtain a mixed solution;

[0129] (3) Sterilization: The mixture is sterilized at a temperature of 115℃ and a pressure of 0.5Mpa for 30 minutes to obtain the culture medium.

[0130] The method for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-tolerant properties using the culture medium includes the following steps:

[0131] (1) Collect sugarcane rhizosphere soil and prepare sugarcane rhizosphere soil suspension;

[0132] (2) The sugarcane rhizosphere soil suspension was placed in the culture medium and cultured to form colonies, and the strain was obtained;

[0133] (3) The strain was purified and identified, and it was found that the acid-aluminum-resistant nitrogen-fixing bacteria could not be screened out through the culture medium.

[0134] Comparative Example 3

[0135] Simultaneously, a culture medium for screening nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria was developed. The culture medium consisted of: 15 g / L agar, 8 g / L glucose, 3 g / L sucrose, 4 g / L peptone, 1 g / L soluble starch, 0.5 g / L urea, 1.2 g / L sodium chloride, 0.4 g / L magnesium sulfate, 1.3 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, 0.1 g / L aluminum chloride, 0.1 g / L manganese chloride, 0.1 g / L sodium molybdate, 2.45 g / L activated carbon powder, 38 ml / L spinach juice, 43 ml / L rapeseed juice, and 0.5 g / L quinoa flour. The pH was controlled at 5.8, with the remainder being water. The spinach juice and rapeseed juice were freshly squeezed.

[0136] The pH value of the culture medium is controlled using citric acid and sodium citrate.

[0137] The spinach juice contains 80-100 mg / kg of aluminum, and the rapeseed juice contains 60-80 mg / kg of aluminum.

[0138] A method for preparing a culture medium for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-resistant properties includes the following steps:

[0139] (1) Preparation of spinach juice and rapeseed juice:

[0140] 1.1) Select fresh spinach with an aluminum content of about 120 mg / kg, cut off the roots, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain spinach juice with an aluminum content of 80-100 mg / kg.

[0141] 1.2) Select fresh rapeseed with an aluminum content of about 100 mg / kg, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain rapeseed juice with an aluminum content of 60-80 mg / kg.

[0142] (2) Weighing: Weigh each raw material that makes up the culture medium according to the ratio;

[0143] (3) Mixing and preparation: Mix all the raw materials that make up the culture medium evenly, and use citric acid and sodium citrate to control the pH value at 5.8 to obtain a mixed solution;

[0144] (4) Sterilization: The mixture is sterilized at a temperature of 115℃ and a pressure of 0.5Mpa for 30 minutes to obtain the culture medium.

[0145] The method for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-tolerant properties using the culture medium includes the following steps:

[0146] (1) Collect sugarcane rhizosphere soil and prepare sugarcane rhizosphere soil suspension;

[0147] (2) The sugarcane rhizosphere soil suspension was placed in the culture medium and cultured to form colonies, and the strain was obtained;

[0148] (3) The strain was purified and identified, and it was found that the acid-aluminum-resistant nitrogen-fixing bacteria could not be screened out through the culture medium.

[0149] Comparative Example 4

[0150] Simultaneously, a culture medium for screening nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria was developed. The culture medium consisted of: 15 g / L agar, 8 g / L glucose, 3 g / L sucrose, 4 g / L peptone, 1 g / L soluble starch, 0.5 g / L urea, 1.2 g / L sodium chloride, 0.4 g / L magnesium sulfate, 1.3 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, 0.1 g / L aluminum chloride, 0.1 g / L manganese chloride, 0.1 g / L sodium molybdate, 2.45 g / L bagasse powder, 38 ml / L spinach juice, 43 ml / L rapeseed juice, and 0.5 g / L quinoa flour. The pH was controlled at 5.8, with the remainder being water. The spinach juice and rapeseed juice were both freshly squeezed.

[0151] The pH value of the culture medium is controlled using citric acid and sodium citrate.

[0152] The spinach juice contains 80-100 mg / kg of aluminum, and the rapeseed juice contains 60-80 mg / kg of aluminum.

[0153] A method for preparing a culture medium for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-resistant properties includes the following steps:

[0154] (1) Preparation of spinach juice and rapeseed juice:

[0155] 1.1) Select fresh spinach with an aluminum content of about 120 mg / kg, cut off the roots, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain spinach juice with an aluminum content of 80-100 mg / kg.

[0156] 1.2) Select fresh rapeseed with an aluminum content of about 100 mg / kg, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain rapeseed juice with an aluminum content of 60-80 mg / kg.

[0157] (2) Weighing: Weigh each raw material that makes up the culture medium according to the ratio;

[0158] (3) Mixing and preparation: Mix all the raw materials that make up the culture medium evenly, and use citric acid and sodium citrate to control the pH value at 5.8 to obtain a mixed solution;

[0159] (4) Sterilization: The mixture is sterilized at a temperature of 115℃ and a pressure of 0.5Mpa for 30 minutes to obtain the culture medium.

[0160] The method for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-tolerant properties using the culture medium includes the following steps:

[0161] (1) Collect sugarcane rhizosphere soil and prepare sugarcane rhizosphere soil suspension;

[0162] (2) The sugarcane rhizosphere soil suspension was placed in the culture medium and cultured to form colonies, and the strain was obtained;

[0163] (3) The strain was purified and identified, and it was found that the acid-aluminum-resistant nitrogen-fixing bacteria could not be screened out through the culture medium.

[0164] Comparative Example 5

[0165] Simultaneously, a culture medium for screening nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria was developed. The culture medium consisted of: 15 g / L agar, 8 g / L glucose, 3 g / L sucrose, 4 g / L peptone, 1 g / L soluble starch, 0.5 g / L urea, 1.2 g / L sodium chloride, 0.4 g / L magnesium sulfate, 1.3 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, 0.1 g / L aluminum chloride, 0.1 g / L manganese chloride, 0.1 g / L sodium molybdate, 2.45 g / L sugarcane leaf powder, 38 ml / L spinach juice, 43 ml / L rapeseed juice, and 0.5 g / L quinoa flour. The pH was controlled at 5.8, with the remainder being water. The spinach juice and rapeseed juice were both freshly squeezed.

[0166] The pH value of the culture medium is controlled using citric acid and sodium citrate.

[0167] The spinach juice contains 80-100 mg / kg of aluminum, and the rapeseed juice contains 60-80 mg / kg of aluminum.

[0168] A method for preparing a culture medium for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-resistant properties includes the following steps:

[0169] (1) Preparation of spinach juice and rapeseed juice:

[0170] 1.1) Select fresh spinach with an aluminum content of about 120 mg / kg, cut off the roots, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain spinach juice with an aluminum content of 80-100 mg / kg.

[0171] 1.2) Select fresh rapeseed with an aluminum content of about 100 mg / kg, wash it clean, press it into juice, filter it with a 0.22 μm sterile filter membrane to obtain rapeseed juice with an aluminum content of 60-80 mg / kg.

[0172] (2) Weighing: Weigh each raw material that makes up the culture medium according to the ratio;

[0173] (3) Mixing and preparation: Mix all the raw materials that make up the culture medium evenly, and use citric acid and sodium citrate to control the pH value at 5.8 to obtain a mixed solution;

[0174] (4) Sterilization: The mixture is sterilized at a temperature of 115℃ and a pressure of 0.5Mpa for 30 minutes to obtain the culture medium.

[0175] The method for simultaneously screening nitrogen-fixing bacteria with nitrogen-fixing and aluminum-tolerant properties using the culture medium includes the following steps:

[0176] (1) Collect sugarcane rhizosphere soil and prepare sugarcane rhizosphere soil suspension;

[0177] (2) The sugarcane rhizosphere soil suspension was placed in the culture medium and cultured to form colonies, and the strain was obtained;

[0178] (3) The strain was purified and identified, and it was found that the acid-aluminum-resistant nitrogen-fixing bacteria could not be screened out through the culture medium.

[0179] The separation and screening results of Examples 1-5 and Comparative Examples 1-5 were statistically analyzed, and the statistical results are shown in Table 1 below.

[0180] Table 1

[0181]

[0182] The total aluminum content of the screened aluminum-resistant nitrogen-fixing bacteria was determined. The results showed that the highest total aluminum content of the screened aluminum-resistant nitrogen-fixing bacteria reached 5000–5500 μg / g. In the above statistical results, the nitrogen content of both the nitrogen-fixing bacteria and the aluminum-resistant nitrogen-fixing bacteria was uniformly controlled at 2.0 x 10⁻⁶. 5Therefore, it can be seen that using aluminum chloride, spinach juice, and rapeseed juice to simulate an acidic aluminum environment, with a lower pH value, provides abundant aluminum, micronutrients, physiologically active substances, and growth hormones. This acidic aluminum environment does not have a toxic effect on nitrogen-fixing bacteria, allowing for the simultaneous screening of nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria. The highest total aluminum content tolerance of the screened aluminum-tolerant nitrogen-fixing bacteria reaches 5000-5500 μg / g. Adding appropriate amounts of activated carbon powder, sugarcane bagasse powder, and sugarcane leaf powder can effectively improve the aeration of the agar medium and effectively adsorb the metabolic waste excreted by nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria, avoiding the toxic effects of metabolic waste on these bacteria. This facilitates the simultaneous screening of nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria and significantly improves their yield. The addition of an appropriate amount of quinoa powder to the culture medium can also enhance the nitrogen-fixing capacity of the nitrogen-fixing bacteria.

[0183] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A culture medium for simultaneously screening nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria, characterized in that, The culture medium consists of: 15 g / L agar, 8 g / L glucose, 3 g / L sucrose, 4 g / L peptone, 1 g / L soluble starch, 0.5 g / L urea, 1.2 g / L sodium chloride, 0.4 g / L magnesium sulfate, 1.3 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, 0.1 g / L aluminum chloride, 0.1 g / L manganese chloride, 0.1 g / L sodium molybdate, 1.2–1.5 g / L activated carbon powder, 0.4–0.7 g / L bagasse powder, 0.3–0.5 g / L sugarcane leaf powder, 35–40 ml / L spinach juice, and 40–45 ml / L rapeseed juice, with the pH controlled at 5.5–6.5, and the remainder being water. The pH value of the culture medium is controlled using citric acid and sodium citrate. The spinach juice contains 80-100 mg / kg of aluminum, and the rapeseed juice contains 60-80 mg / kg of aluminum. The method for preparing the spinach juice is as follows: Select fresh spinach with an aluminum content of about 120 mg / kg, cut off the roots, wash it clean, press it into juice, filter it, and obtain spinach juice with an aluminum content of 80-100 mg / kg. The method for preparing the rapeseed juice is as follows: Select fresh rapeseed with an aluminum content of about 100 mg / kg, wash it clean, press it into juice, filter it, and obtain rapeseed juice with an aluminum content of 60-80 mg / kg.

2. The culture medium for simultaneously screening nitrogen-fixing and aluminum-resistant nitrogen-fixing bacteria according to claim 1, characterized in that: The culture medium consists of: 15 g / L agar, 8 g / L glucose, 3 g / L sucrose, 4 g / L peptone, 1 g / L soluble starch, 0.5 g / L urea, 1.2 g / L sodium chloride, 0.4 g / L magnesium sulfate, 1.3 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, 0.1 g / L aluminum chloride, 0.1 g / L manganese chloride, 0.1 g / L sodium molybdate, 1.3–1.5 g / L activated carbon powder, 0.5–0.7 g / L bagasse powder, 0.4–0.5 g / L sugarcane leaf powder, 37–40 ml / L spinach juice, and 42–45 ml / L rapeseed juice, with the pH controlled at 5.5–6.5, and the remainder being water.

3. The culture medium for simultaneously screening nitrogen-fixing and aluminum-tolerant nitrogen-fixing bacteria according to claim 1, characterized in that: The culture medium consists of: 15 g / L agar, 8 g / L glucose, 3 g / L sucrose, 4 g / L peptone, 1 g / L soluble starch, 0.5 g / L urea, 1.2 g / L sodium chloride, 0.4 g / L magnesium sulfate, 1.3 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, 0.1 g / L aluminum chloride, 0.1 g / L manganese chloride, 0.1 g / L sodium molybdate, 1.4 g / L activated carbon powder, 0.6 g / L bagasse powder, 0.45 g / L sugarcane leaf powder, 38 ml / L spinach juice, and 43 ml / L rapeseed juice, with the pH controlled at 5.5–5.8, and the remainder being water.

4. The culture medium for simultaneously screening nitrogen-fixing and aluminum-resistant nitrogen-fixing bacteria according to claim 1, characterized in that: The spinach juice is freshly squeezed spinach juice, and the rapeseed juice is freshly squeezed rapeseed juice.

5. The culture medium for simultaneously screening nitrogen-fixing and acid-aluminum-tolerant nitrogen-fixing bacteria according to claim 1, characterized in that: The culture medium also contains 0.4–0.6 g / L of quinoa powder.

6. The culture medium for simultaneously screening nitrogen-fixing and acid-aluminum-resistant nitrogen-fixing bacteria according to claim 5, characterized in that: The culture medium also contains 0.5 g / L of quinoa powder.

7. A method for preparing a culture medium for simultaneously screening nitrogen-fixing and aluminum-resistant nitrogen-fixing bacteria according to any one of claims 1-6, characterized in that: Includes the following steps: (1) Preparation of spinach juice and rapeseed juice; (2) Weighing: Weigh each raw material that makes up the culture medium according to the ratio; (3) Mixing and preparation: Mix all the raw materials that make up the culture medium evenly, and use citric acid and sodium citrate to control the pH value at 5.5 to 5.8 to obtain a mixed solution; (4) Sterilization: The mixture is sterilized to obtain the culture medium.

8. The method for preparing a culture medium for simultaneously screening nitrogen-fixing and aluminum-resistant nitrogen-fixing bacteria according to claim 7, characterized in that: The filter membrane used in step (1) to prepare spinach juice and rapeseed juice is a 0.22μm sterile filter membrane.

9. The method for preparing a culture medium for simultaneously screening nitrogen-fixing and aluminum-resistant nitrogen-fixing bacteria according to claim 7, characterized in that: In step (4), the mixture is sterilized at a temperature of 115°C and a pressure of 0.5 MPa for 30 minutes to obtain the culture medium.

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