A modified graphene-immobilized functional strain and its application

Abstract

This invention belongs to the field of microbial fertilizer and bioengineering technology, and discloses a modified graphene-immobilized functional strain and its applications. It includes optimizing the selection and preparation of the culture medium, strain activation and modified graphene immobilization, and identification of the binding effect. This invention achieves dual cost reduction, is suitable for industrial production, synergistically improves immobilization efficiency and strain stability, dually enhances strain activity and metabolic function, is environmentally friendly, and expands application scenarios.

Description

Technical Field

[0001] This invention belongs to the field of microbial fertilizer and bioengineering technology, specifically relating to a modified graphene-immobilized functional strain and its application. Background Technology

[0002] Traditional microbial fertilizers face key challenges in agricultural applications, including low strain survival rates, limited functionality, and insufficient carrier performance. During storage and in the soil environment, strains are susceptible to stresses such as pH, temperature, and salt ions, leading to activity decline and difficulty in maintaining effective bacterial counts. For example, after long-term storage, the survival rate of strains in traditional liquid microbial agents or organic fertilizer mixtures often drops below 60% due to environmental stress. Furthermore, while existing strains such as Bacillus subtilis and phosphate-solubilizing bacteria can improve soil microecology, their low metabolic product release efficiency prevents them from continuously providing crops with nutrients such as amino acids, enzymes, and growth hormones, thus limiting the fertilizer's growth-promoting effect.

[0003] Furthermore, commonly used carriers such as sodium alginate and vermiculite have limited adsorption capacity, lack the effect of promoting the metabolic activity of bacterial strains, are difficult to stably load bacterial strains, and are prone to loss; while modified graphene has high adsorption performance (theoretical specific surface area reaches 2630 m²). 2 With unique advantages such as high conductivity, thermal conductivity, and environmental tolerance, it can stabilize the loaded strain through physical adsorption and chemical conjugation. Its electronic conduction ability may also regulate the electronic metabolic pathway of the strain to promote enzyme activity and secondary metabolite synthesis. In the prior art, such as the Chinese invention patent with patent application number 202510892111.6, the phosphate rock thermal activation product crosslinking carrier is prepared by phosphate rock thermal activation product + crosslinking agent. It can provide a three-dimensional network porous loading space, naturally contains slow-release nutrients such as phosphorus, calcium, and magnesium, and can be adapted to traditional mineral processing equipment. It is suitable for low-cost field planting and phosphorus-deficient soil improvement. However, it has the problems of small specific surface area, easy detachment due to physical fixation, and lack of heavy metal chelation and strain metabolic enhancement capabilities. Furthermore, traditional culture media (such as NA, YPD, and PDA) are not only expensive, but also produce strains with low activity. Their carbon source is often glucose (market price 151.80 yuan / kg), resulting in high raw material costs. Additionally, they lack adaptive design for the combination of strains and functional carriers, failing to provide a high-quality culture environment for the formation of modified graphene-strain complexes, further restricting the improvement of fertilizer efficiency. Existing technologies, such as the Chinese invention patent with patent application number 202410746694.7, use traditional compound fertilizers + graphene chelating agents + microbial assistance to prepare compound fertilizers. This can improve soil quality, increase soil clay content, enhance soil's ability to retain nutrients, and reduce nutrient loss, making it suitable for scenarios with high demand for fast-acting fertilizers and poor soil physical properties. However, it suffers from high costs, weak microbial stability, and complex processes. How to utilize low-cost optimized culture media for bacterial cultivation, and combine the bacterial strains with modified graphene carriers to achieve integrated high-efficiency cultivation, stable fixation, and functional enhancement of the strains, while making them more suitable for ecological agriculture, facility agriculture, and planting in extreme environments (such as saline-alkali land), remains a key issue restricting the industrialization of this technology. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a modified graphene-immobilized functional strain and its application. Through a synergistic approach of "optimized culture medium cultivation + modified graphene immobilization", it solves the core problems of low strain survival rate, insufficient metabolic function, poor environmental tolerance and high cultivation cost in traditional microbial fertilizers, and achieves efficient colonization, continuous metabolism and stress resistance enhancement of the strain in agricultural applications.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution: a modified graphene-immobilized functional strain, the preparation steps of which include: 1. Optimization of culture medium selection and preparation: A low-cost optimized culture medium suitable for culturing fungi and bacteria was used as the basis for strain culture. The formula was as follows: molasses 10.0-20.0 g / L, peptone 5.0-15.0 g / L, yeast extract 5.0-8.0 g / L, magnesium sulfate heptahydrate 0.02-0.5 g / L, modified graphene 0.25-0.5 g / L, with the balance being sterile water; Preparation steps: Weigh each component according to the formula ratio, mix and heat to dissolve, adjust the pH value to 7.0-7.2 with 1 mol / L food-grade NaOH solution, and sterilize at 115 ℃ for 30 min to obtain the optimized culture medium. This culture medium is suitable for a variety of functional strains and can enhance the activity of strains. Compared with the traditional LB medium, the OD600 value of the strains can be increased by up to 5-7 times, providing a source of highly active strains for the immobilization of modified graphene. 2. Strain activation and modified graphene immobilization: Strains were selected from soybean rhizosphere soil samples. After serial dilution, the samples were initially screened using phosphorus / potassium solubilization and growth-promoting media to select candidate colonies with large phosphorus / potassium solubilization zones and high IAA secretion. Then, Pseudomonas and Gynostemma pentaphyllum were selected for identification by combining 16S rDNA sequencing to select Pseudomonas and Gynostemma pentaphyllum with good nitrogen fixation, phosphorus / potassium solubilization and growth-promoting abilities. Directional fixation: Add modified graphene dry powder to the optimized culture medium cultured to the target OD600 value in step 1, set different co-culture time groups, and utilize the high specific surface area and surface functional groups of modified graphene to bind with the surface components of the strain. After the culture is completed, freeze-dry the modified graphene-strain complex and use it directly as a microbial agent. 3. Binding effect assessment: The binding effect was verified using scanning electron microscopy (SEM), attenuated total reflectance mode Fourier transform infrared spectroscopy (ATR-FTIR), and powder X-ray diffraction (XRD). SEM observation: The strains were uniformly loaded onto the surface of the modified graphene, forming a composite layer with a thickness of 50-100 nm, with no obvious aggregation. ATR-FTIR analysis: The NH peak of the bacterial community in the composite system shifted by 30 cm⁻¹ -1 The C=C peak full width at half maximum (FWHM) of modified graphene increased by 73%, and an additional 1265 cm⁻¹ was added. -1 The ether bond peaks demonstrate the combination of hydrogen bonds, π-π conjugation, and ether bonds, without disrupting the core structure of either. XRD analysis: The main peak intensity of modified graphene at 26.5 ° in group G5 was moderate (268), with a highly symmetrical peak shape and reasonable broadening (25%). The background at low angles increased steadily, which is consistent with the signal superposition law of the "modified graphene + bacteria + optimized culture medium" composite system, and there was no interference from impurity peaks.

[0006] Furthermore, in step 1, the sterile water in the culture medium is optimized to be distilled water or purified water.

[0007] Furthermore, in step 2, the amount of modified graphene dry powder added in the orientation fixation step is 0.05 wt%, and the different co-cultivation time groups are: 0 h, 12 h, 24 h, 36 h, and 48 h.

[0008] Furthermore, the infrared transmittance test range during step 3, ATR-FTIR analysis, is 4000 ~ 650 cm⁻¹. -1 Scan rate: 10 kHz, Accuracy: 4 cm -1 .

[0009] Furthermore, in step 3 of the XRD analysis process, the radiation source is Cu K radiation, the accelerating voltage is 45 kV, the transmission current is 40 mA, the diffraction angle (2θ) measurement range is 10°~60°, and the scanning speed is 3 degrees / minute.

[0010] Another objective of this invention is to protect the application of the above-mentioned modified graphene-immobilized functional strains, specifically in the field of agricultural planting.

[0011] Furthermore, the modified graphene-immobilized functional strains can be applied to basic food crops (corn, wheat, etc.), cash crops (soybeans, peanuts, rapeseed, etc.), vegetables and fruits (broccoli, tomatoes, cucumbers, etc.), and medicinal plants (Camptotheca acuminata, Astragalus membranaceus, etc.). Due to their strong stress resistance and wide adaptability, they can be used for ecological restoration plants in saline-alkali land (Haloxylon ammodendron, Hippophae rhamnoides, etc.) and other stress-resistant crops (millet, oats, etc.). For example, they can be used for the cultivation of soybean seedlings.

[0012] The advantages of this invention compared to the prior art are: 1) Dual cost reduction, suitable for industrial production Culture medium cost: By using molasses instead of glucose as a carbon source, the raw material cost is reduced by 17 times. At the same time, the expensive nutrient additives in traditional culture media are eliminated, resulting in an overall reduction of culture medium cost of more than 60%.

[0013] Fixed process cost: Relying on the high activity of the optimized culture medium, the consumption of additional immobilization reagents (such as glutaraldehyde) is eliminated, the process flow is simplified, energy consumption is reduced, and it can be directly adapted to existing microbial fermentation equipment without the need to modify the production line.

[0014] Synergistic improvement in fixation efficiency and strain stability High fixation efficiency: modified graphene ≥300m 2 The high specific surface area of ​​ / g provides abundant adsorption sites. Combined with highly active strains cultivated in optimized culture media, the immobilization effect with the carrier is significant, enabling a stable binding between the strain and the carrier.

[0015] Strong stability: The dual mechanisms of physical and chemical adsorption enhance the binding force, and the survival rate of the strain is significantly improved compared with that of free strains in low-temperature storage and soil application scenarios. When faced with complex stress environments such as pH fluctuations, high salt, and heavy metals, modified graphene can provide effective protection for the strains and help them maintain a high survival rate.

[0016] Dual enhancement of strain activity and metabolic function Enhanced activity: Optimized culture medium can increase the OD of the strain. 600The value is 5-7 times higher than that of LB medium, providing a basis for highly active strains for immobilization; modified graphene can affect the metabolism of strains by regulating electron conduction characteristics, thereby promoting the synthesis of functional products such as indoleacetic acid and acid phosphatase, resulting in a significant increase in product yield compared to free strains.

[0017] Agricultural efficacy: The functional strains carried by the compound microbial agent have potassium and phosphorus solubilizing properties, which can specifically activate the insoluble phosphorus and potassium nutrients in the soil, helping crops to absorb and utilize them. At the same time, it can significantly promote crop root growth and increase root fresh weight. It is also compatible with a variety of functional strains such as phosphorus-solubilizing bacteria and nitrogen-fixing bacteria, and is suitable for the growth needs of different crops such as wheat and tomatoes, effectively making up for the shortcomings of traditional microbial fertilizers in terms of single function and limited efficacy.

[0018] Environmental friendliness and application scenario expansion Green and safe: The optimized culture medium contains no toxic components, and the modified graphene can be gradually degraded through natural oxidation (half-life ≤ 12 months), avoiding the use of toxic chemical cross-linking agents and eliminating the risk of secondary pollution; after application, it can also increase the organic matter content of the soil and improve the soil micro-ecology.

[0019] Wide range of applications: It is not only applicable to the field of agricultural fertilizers, but can also be extended to sewage purification, biodegradation and other scenarios. It is especially suitable for industrial needs such as soil improvement in dry red wine producing areas and planting of crops in facility agriculture. It is more versatile than traditional single-function carrier / culture medium systems.

[0020] Differentiated innovation from existing technologies: 1) “Culture medium-carrier” synergistic design: For the first time, optimized culture medium for culturing fungi and bacteria is combined with modified graphene immobilization technology, breaking through the traditional separation mode of “culturing first and then immobilizing”. By utilizing the high activity of the optimized culture medium and the immobilization advantage of modified graphene, “culturing-immobilization” integration is achieved, shortening the process cycle by more than 50%.

[0021] 2) Triple synergy of functions: Modified graphene not only serves as a physical carrier but also regulates the metabolism of the strain; optimized culture medium not only provides nutrition but also enhances the binding compatibility between the strain and the carrier; the strain simultaneously possesses growth-promoting functions and multi-environment adaptability. The three synergistically achieve the triple effect of "high-efficiency cultivation, stable fixation, and enhanced function".

[0022] 3) Cost and performance balance: By selecting molasses carbon source and modified graphene, the cost can be reduced while ensuring that the activity of the strain, fixation efficiency and the yield of metabolites do not decrease, thus solving the contradiction of "low cost inevitably leads to low performance" in traditional technologies. Attached Figure Description

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Figure 1A graph showing the nitrogen fixation, phosphorus / potassium solubilization, and IAA production capabilities of the selected bacterial communities ("+" indicates strong capability, "-" indicates no capability); Figure 2 This is a schematic diagram illustrating the effect of different basal culture medium formulations on the OD600 values ​​of different strains. Figure 3 A schematic diagram showing the effect of optimized culture medium and control culture medium on the OD600 values ​​of different strains at different time points; Figure 4 Schematic diagram of SEM identification of modified graphene combined with bacterial community, where (a) modified graphene aqueous solution; (b) modified graphene + culture medium; (c) bacterial community + culture medium; (d) modified graphene + bacterial community + culture medium (0h); (e) modified graphene + bacterial community + culture medium (12h); (f) modified graphene + bacterial community + culture medium (24h); (g) modified graphene + bacterial community + culture medium (36h); (h) modified graphene + bacterial community + culture medium (48h). Figure 5 Schematic diagram of ATR-FTIR identification of modified graphene-bound bacterial communities; Figure 6 Schematic diagram of XRD identification of modified graphene-bound bacterial communities; Figure 7 A schematic diagram showing the results of soybean cultivation using modified graphene compound fertilizer. Detailed Implementation

[0024] The present invention is described in detail below through specific embodiments, but this does not limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained commercially.

[0025] Example 1 Optimize culture medium The following strains were cultured using the following culture media: fungi G2-12, C3-2, C3-1, C3-8 and bacteria C4-1.

[0026] The culture medium formulation is as follows: 10.0 g / L peptone, 5 g / L yeast extract, 10.0 g / L sodium chloride, with the remainder being sterile water. The pH of the culture medium is adjusted to 7.0 using 1 mol / L food-grade NaOH solution. 7.20, sterilized at 115℃ for 30 min, served as the control culture medium.

[0027] Culture method: Inoculation amount 2%, 30℃, 150 r / min, shake culture for 18 h.

[0028] Example 2 Comparative Experiment of Basic Culture Medium Formulation in Comparative Example 1 Formula 1-1: 20.0 g / L glucose, 10.0 g / L peptone, 5.0 g / L yeast extract, 1.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.5 g / L modified graphene, and the balance is sterile water.

[0029] Formula 1-2: 20.0 g / L glucose, 10.0 g / L peptone, 5.0 g / L yeast extract, 1.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.25 g / L modified graphene, and the balance is sterile water.

[0030] The basal culture media were prepared according to formulas 1-1 and 1-2, respectively, and the above five strains were cultured under the same culture conditions as in Example 1. After 18 h of shaking culture, the OD of the bacterial suspension was measured. 600 Value, can be obtained from Figure 2 The results showed that the activity of the strains was superior to that of the control group.

[0031] Example 3 Comparative Experiment of Basic Culture Medium Formulation in Comparative Example 1 Formula 2-1: Molasses 10.0 g / L, Peptone 10.0 g / L, Yeast extract 5.0 g / L, Potassium dihydrogen phosphate 1.0 g / L, Magnesium sulfate heptahydrate 0.5 g / L, Modified graphene 0.5 g / L, Balance: Sterile water. Formula 2-2: Molasses 15.0 g / L, Peptone 10.0 g / L, Yeast extract 5.0 g / L, Potassium dihydrogen phosphate 1.0 g / L, Magnesium sulfate heptahydrate 0.5 g / L, Modified graphene 0.5 g / L, Balance: Sterile water.

[0032] Formula 2-3: Molasses 20.0 g / L, Peptone 10.0 g / L, Yeast extract 5.0 g / L, Potassium dihydrogen phosphate 1.0 g / L, Magnesium sulfate heptahydrate 0.5 g / L, Modified graphene 0.5 g / L, Balance: Sterile water.

[0033] Formulas 2-4: Molasses 10.0 g / L, Peptone 10.0 g / L, Yeast Extract 5.0 g / L, Potassium Dihydrogen Phosphate 1.0 g / L, Magnesium Sulfate Heptahydrate 0.5 g / L, Modified Graphene 0.25 g / L, Balance: Sterile Water.

[0034] Formulas 2-5: Molasses 15.0 g / L, Peptone 10.0 g / L, Yeast extract 5.0 g / L, Potassium dihydrogen phosphate 1.0 g / L, Magnesium sulfate heptahydrate 0.5 g / L, Modified graphene 0.25 g / L, Balance: Sterile water.

[0035] Formulas 2-6: Molasses 20.0 g / L, Peptone 10.0 g / L, Yeast Extract 5.0 g / L, Potassium Dihydrogen Phosphate 1.0 g / L, Magnesium Sulfate Heptahydrate 0.5 g / L, Modified Graphene 0.25 g / L, Balance: Sterile Water.

[0036] Basic culture media were prepared according to formulations 2-1, 2-2, 2-3, 2-4, 2-5, and 2-6, respectively, and the above five strains were cultured under the same conditions as in Example 1. After 18 hours of shaking culture, the OD of the bacterial suspension was measured. 600 Value, can be obtained from Figure 2 As can be seen, the activity of the strains was superior to that of the control group. Example 4 Comparative Experiment of Basic Culture Medium Formula 3-1 (Comparative Example 1): Glucose 20.0 g / L, soybean meal powder 5.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate heptahydrate 0.5 g / L, modified graphene 0.5 g / L, balance sterile water.

[0037] Formula 3-2: 20.0 g / L glucose, 10.0 g / L soybean meal, 1.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.5 g / L modified graphene, and the balance is sterile water.

[0038] Formula 3-3: 20.0 g / L glucose, 15.0 g / L soybean meal, 1.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.5 g / L modified graphene, and the balance is sterile water.

[0039] Formula 3-4: 20.0 g / L glucose, 5.0 g / L soybean meal, 1.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.25 g / L modified graphene, and the balance is sterile water.

[0040] Formulas 3-5: 20.0 g / L glucose, 10.0 g / L soybean meal, 1.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.25 g / L modified graphene, with the balance being sterile water.

[0041] Formulas 3-6: 20.0 g / L glucose, 15.0 g / L soybean meal, 1.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.25 g / L modified graphene, with the balance being sterile water.

[0042] Basic culture media were prepared according to formulations 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, respectively, and the above five strains were cultured under the same conditions as in Example 1. After 18 h of shaking culture, the OD of the bacterial suspension was measured. 600 Value, can be obtained from Figure 2As can be seen, the activity of the strains was superior to that of the control group. Example 5 Comparative Experiment of Basic Culture Medium Formulation in Comparative Example 1 Formula 4-1: 20.0 g / L glucose, 10.0 g / L peptone, 5.0 g / L yeast extract, 0.005 g / L ferric chloride, 0.5 g / L modified graphene, and the balance is sterile water.

[0043] Formula 4-2: 20.0 g / L glucose, 10.0 g / L peptone, 5.0 g / L yeast extract, 0.01 g / L ferric chloride, 0.5 g / L modified graphene, and the balance is sterile water.

[0044] Formula 4-3: 20.0 g / L glucose, 10.0 g / L peptone, 5.0 g / L yeast extract, 0.015 g / L ferric chloride, 0.5 g / L modified graphene, and the balance is sterile water.

[0045] Formula 4-4: Glucose 20.0 g / L, Peptone 10.0 g / L, Yeast extract 5.0 g / L, Ferric chloride 0.005 g / L, Modified graphene 0.25 g / L, Balance: Sterile water.

[0046] Formula 4-5: 20.0 g / L glucose, 10.0 g / L peptone, 5.0 g / L yeast extract, 0.01 g / L ferric chloride, 0.25 g / L modified graphene, and the balance is sterile water.

[0047] Formulas 4-6: 20.0 g / L glucose, 10.0 g / L peptone, 5.0 g / L yeast extract, 0.015 g / L ferric chloride, 0.25 g / L modified graphene, and the balance is sterile water.

[0048] Basic culture media were prepared according to formulations 4-1, 4-2, 4-3, 4-4, 4-5, and 4-6, respectively, and the above five strains were cultured under the same culture conditions as in Example 1. After 18 h of shaking culture, the OD of the bacterial suspension was measured. 600 Value, can be obtained from Figure 2 The results showed that the activity of the strains was superior to that of the control group.

[0049] Example 6 Comparative Experiment of Basic Culture Medium Formulation in Comparative Example 1 Formula 5-1: Glucose 20.0 g / L, Peptone 10.0 g / L, Yeast extract 5.0 g / L, Vitamin B1 0.005 g / L, Modified graphene 0.5 g / L, Balance: Sterile water.

[0050] Formula 5-2: Glucose 20.0 g / L, Peptone 10.0 g / L, Yeast extract 5.0 g / L, Vitamin B1 0.01 g / L, Modified graphene 0.5 g / L, Balance: Sterile water.

[0051] Formula 5-3: Glucose 20.0 g / L, Peptone 10.0 g / L, Yeast extract 5.0 g / L, Vitamin B1 0.015 g / L, Modified graphene 0.5 g / L, Balance: Sterile water.

[0052] Formula 5-4: Glucose 20.0 g / L, Peptone 10.0 g / L, Yeast extract 5.0 g / L, Vitamin B1 0.005 g / L, Modified graphene 0.25 g / L, Balance: Sterile water.

[0053] Formula 5-5: Glucose 20.0 g / L, Peptone 10.0 g / L, Yeast extract 5.0 g / L, Vitamin B1 0.01 g / L, Modified graphene 0.25 g / L, Balance: Sterile water.

[0054] Formula 5-6: Glucose 20.0 g / L, Peptone 10.0 g / L, Yeast extract 5.0 g / L, Vitamin B1 0.015 g / L, Modified graphene 0.25 g / L, Balance: Sterile water.

[0055] Basic culture media were prepared according to formulations 5-1, 5-2, 5-3, 5-4, 5-5, and 5-6, respectively, and the above five strains were cultured under the same conditions as in Example 1. After 18 hours of shaking culture, the OD of the bacterial suspension was measured. 600 Value, can be obtained from Figure 2 The results showed that the activity of the strains was superior to that of the control group.

[0056] from Figure 2 As can be seen, the activity of the strains in the basic culture medium prepared by this invention was significantly improved after the beneficial substances were combined, especially Formula 2-2, whose activity increased by about 5-7 times compared with the control group.

[0057] Example 7: In addition to the above examples, this invention detected the OD values ​​of bacterial suspensions of these five strains at different time points. 600 The values ​​were 0 h, 2 h, 6 h, 12 h, 18 h, and 24 h, respectively. Regardless of whether the LB medium reached its maximum value at 24 hours or the optimized medium reached its maximum value at 18 hours, such as... Figure 3 As shown.

[0058] Example 8 The modified graphene powder was sterilized and ready for use. Modified graphene water-based powder was added to sterile water to obtain modified graphene aqueous solution (concentration of 0.05%), which was then freeze-dried to obtain sample CK1.

[0059] Example 9 After preparing the optimized culture medium without modified graphene in formulation 2-2 of Example 3, sterilize it, add modified graphene dry powder (the amount added is the same as in Example 8) to the optimized culture medium, freeze dry, and obtain sample CK2.

[0060] Example 10 Five functional bacterial strains were inoculated into LB medium and activated by shaking culture at 28°C for 10 h. Then, the five strains were added to the same culture medium without modified graphene (Formula 2-2 in Example 3) and cultured for approximately 10 h to achieve OD. 600 =1, freeze-dried, to obtain sample CK3.

[0061] Example 11 Five functional bacterial strains were inoculated into LB medium and activated by shaking culture at 28°C for 10 h. Then, the five strains were added to the same culture medium without modified graphene (Formula 2-2 in Example 3) and cultured for approximately 10 h to achieve OD. 600 =1, add modified graphene dry powder (the amount added is the same as in Example 8) to the optimized culture medium, set the co-culture time to 0 h, freeze dry, and obtain sample G1.

[0062] Example 12 Five functional bacterial strains were inoculated into LB medium and activated by shaking culture at 28°C for 10 h. Then, the five strains were added to the same culture medium without modified graphene (Formula 2-2 in Example 3) and cultured for approximately 10 h to achieve OD. 600 =1, add modified graphene powder (the amount added is the same as in Example 8) to the optimized culture medium, set the co-culture time to 12 h, freeze dry, and obtain sample G2.

[0063] Example 13 Five functional bacterial strains were inoculated into LB medium and activated by shaking culture at 28°C for 10 h. Then, the five strains were added to the same culture medium without modified graphene (Formula 2-2 in Example 3) and cultured for approximately 10 h to achieve OD. 600 =1, add modified graphene dry powder (the amount added is the same as in Example 8) to the optimized culture medium, set the co-culture time to 24 h, freeze dry, and obtain sample G3.

[0064] Example 14 Five functional bacterial strains were inoculated into LB medium and activated by shaking culture at 28°C for 10 h. Then, the five strains were added to the same culture medium without modified graphene (Formula 2-2 in Example 3) and cultured for approximately 10 h to achieve OD. 600 =1, add modified graphene dry powder (the amount added is the same as in Example 8) to the optimized culture medium, set the co-culture time to 36 h, freeze dry, and obtain sample G4.

[0065] Example 15 Five functional bacterial strains were inoculated into LB medium and activated by shaking culture at 28°C for 10 h. Then, the five strains were added to the same culture medium without modified graphene (Formula 2-2 in Example 3) and cultured for approximately 10 h to achieve OD. 600 =1, add modified graphene dry powder (the amount added is the same as in Example 8) to the optimized culture medium, set the co-culture time to 48 h, freeze dry, and obtain sample G5.

[0066] Effect verification: The modified graphene compound fertilizer prepared in group G5 of Example 8 above was used in soybean cultivation, fertilized once every two weeks, for a total of two applications. The results are as follows... Figure 7 As shown.

[0067] The embodiments described above are merely preferred embodiments of the present invention, and not all feasible embodiments of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A modified graphene-immobilized functional strain, characterized in that, The preparation steps include: S1. Selection and preparation of optimized culture medium: A low-cost optimized culture medium capable of culturing fungi and bacteria was used as the basis for strain culture. The formula was as follows: molasses 10.0-20.0 g / L, peptone 5.0-15.0 g / L, yeast extract 5.0-8.0 g / L, magnesium sulfate heptahydrate 0.02-0.5 g / L, modified graphene 0.25-0.5 g / L, and the balance being sterile water; Preparation steps: Weigh each component according to the formula ratio, mix and heat to dissolve, adjust the pH value to 7.0-7.2 with 1 mol / L food grade NaOH solution, sterilize at 115 ℃ for 30 min to obtain the optimized culture medium; S2. Strain activation and modified graphene immobilization: Strains were selected from soybean rhizosphere soil samples. After serial dilution, the samples were initially screened using phosphorus / potassium solubilization and growth-promoting media to select candidate colonies with large phosphorus / potassium solubilization zones and high IAA secretion. Then, Pseudomonas and Gynostemma pentaphyllum were selected for identification by combining 16S rDNA sequencing to select Pseudomonas and Gynostemma pentaphyllum with good nitrogen fixation, phosphorus / potassium solubilization and growth-promoting abilities. Directional fixation: Add modified graphene dry powder to the optimized culture medium cultured to the target OD600 value in step S1, set different co-culture time groups, and utilize the high specific surface area and surface functional groups of modified graphene to bind with the surface components of the strain. After the culture is completed, freeze-dry the modified graphene-strain complex and use it directly as a microbial agent. S3. Binding effect assessment: The binding effect was verified through SEM observation, ATR-FTIR analysis, and XRD analysis. SEM observation: The strains were uniformly loaded onto the surface of the modified graphene, forming a composite layer with a thickness of 50-100 nm, with no obvious aggregation. ATR-FTIR analysis: The NH peak of the bacterial community in the composite system shifted by 30 cm⁻¹ -1 The C=C peak full width at half maximum (FWHM) of modified graphene increased by 73%, and an additional 1265 cm⁻¹ was added. -1 The ether bond peaks demonstrate the combination of hydrogen bonds, π-π conjugation, and ether bonds, without disrupting the core structure of either. XRD analysis: The main peak intensity of modified graphene at 26.5 ° in group G5 was moderate, with a highly symmetrical peak shape and reasonable broadening. The background at low angles increased steadily, which is consistent with the signal superposition law of the "modified graphene + bacteria + optimized culture medium" composite system, and there was no interference from impurity peaks.

2. The modified graphene-immobilized functional strain according to claim 1, characterized in that, In step S1, the sterile water in the culture medium is optimized to be distilled water or purified water.

3. The modified graphene-immobilized functional strain according to claim 1, characterized in that, In step S2, the amount of modified graphene dry powder added in the orientation fixation step is 0.05 wt%, and the different co-cultivation time groups are: 0 h, 12 h, 24 h, 36 h, and 48 h.

4. The modified graphene-immobilized functional strain according to claim 1, characterized in that, The infrared transmittance test range during step S3ATR-FTIR analysis is 4000 ~ 650 cm⁻¹. -1 Scan rate: 10 kHz, Accuracy: 4 cm -1 .

5. The modified graphene-immobilized functional strain according to claim 1, characterized in that, The radiation source in step S3XRD analysis is Cu K radiation, the accelerating voltage is 45 kV, the transmission current is 40 mA, the diffraction angle (2θ) measurement range is 10°~60°, and the scanning speed is 3 degrees / minute.

6. The application of the modified graphene-immobilized functional strain as described in claim 1, characterized in that, Specifically applied to the field of agricultural planting.

7. The application of the modified graphene-immobilized functional strain according to claim 6, characterized in that, The applications of the modified graphene-immobilized functional strains include use in basic food crops, cash crops, vegetables and fruits, medicinal plants, etc.; and in ecological restoration of saline-alkali land and other stress-resistant crops.

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