Microbial compound fertilizer for treating and improving saline-alkali soil and preparation method of microbial compound fertilizer

Abstract

The invention relates to the technical field of microbial fertilizer preparation, and provides a microbial compound fertilizer for saline-alkali soil treatment and improvement and a preparation method thereof. The method comprises the following steps: co-culturing a plurality of microorganisms in an improved LB culture medium containing sodium chloride, carrying out 10 times of continuous passage domestication, screening out a stable combination, carrying out saline-alkali gradient domestication amplification culture on the screened stable combination, carrying out ultrasonic treatment and centrifugal concentration to obtain a compound bacteria solution, preparing an emulsion from a chitosan solution, the compound bacteria solution and a humic acid mixed solution, and carrying out freeze-drying to obtain the chitosan / humic acid composite bacterial fertilizer. The microbial compound fertilizer for saline-alkali soil treatment and improvement is obtained by carrying out double crosslinking on the raw materials through calcium chloride and glutaraldehyde to form microcapsules, granulating, pre-drying and drying the microcapsules, spraying an outer coating on the microcapsules, and mixing the microcapsules with decomposed sheep manure, monoammonium phosphate and the like. The screened saline-alkali tolerant flora can effectively adapt to saline-alkali stress environment, is high in survival rate, can effectively relieve saline-alkali soil stress, improves the vegetation survival rate and soil quality, and achieves the effect of soil conditioning and improvement.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fertilizer preparation, and particularly relates to a microbial compound fertilizer for saline-alkali soil treatment and improvement and a preparation method thereof. BACKGROUND

[0002] Due to the characteristics of high salinity, high pH value, soil structure compaction and poor nutrient availability, saline-alkali soil has become a key bottleneck restricting the sustainable development of global agriculture. At present, although there are various means for the treatment of saline-alkali soil, they all have significant limitations: chemical improvers can reduce the salinity of saline-alkali soil in the short term, but can easily cause secondary salinization of soil and ecological damage; physical improvement has high cost and is difficult to maintain long-term effects; and conventional microbial fertilizers have the potential of environmental protection and sustainability, but they face multiple challenges in the extreme environment of saline-alkali soil. Specifically, the core functional strains of conventional microbial fertilizers have insufficient stress resistance, and under high salt and high alkali stress, the cell membrane is easily ruptured and the metabolic activity is suddenly reduced, so that the survival rate of live bacteria is usually low within 24 hours after application, and effective colonization cannot be formed. At the same time, due to the lack of synergistic domestication among compound strains, the interspecific antagonism rate is high, resulting in the loss of functional complementarity, such as the inhibition of the growth of nitrogen-fixing bacteria and potassium-dissolving bacteria by the competition for nutrients.

[0003] In addition, the carrier of traditional microbial fertilizers lacks a targeted protective structure, and during storage and application, the strains are easily inactivated due to the impact of temperature, humidity and soil salinity, and the fertilizer particles have poor anti-crushing properties, further reducing the treatment efficiency. Although some technologies attempt to simply domesticate the strains or use embedding methods, the following core problems exist in the existing microbial treatment technology for saline-alkali soil: insufficient stress resistance of strains: conventional microbial strains cannot effectively synthesize osmotic protectants to resist dehydration caused by high osmotic pressure, nor can they maintain intracellular pH homeostasis by activating related transport proteins in the extreme environment of saline-alkali soil with high salt and high pH (pH = 8.5-10.0), resulting in cell membrane rupture, sudden reduction of metabolic activity, low survival rate of live bacteria within a short period after application, and difficulty in forming effective colonization; poor interspecific synergy: compound strains have not been synergistically domesticated, and there is significant antagonism between species, which cannot achieve functional synergy through metabolic complementation, but instead inhibits the growth of each other due to resource competition, resulting in the loss of functions such as nitrogen fixation, phosphorus dissolution and potassium dissolution; low embedding and colonization stability: strains are easily damaged by mechanical shearing and chemical crosslinking during embedding, and lack effective structural protection, so that after being applied to saline-alkali soil, they are difficult to resist the impact of extreme environment and have low colonization rate, and cannot continuously play the improvement role. The activation of stress resistance and metabolism is not synchronized: the existing technology fails to effectively combine the domestication of stress resistance and the activation of metabolism of strains, resulting in low metabolic efficiency of strains with certain stress resistance, which cannot rapidly reproduce and play a function in saline-alkali soil.

[0004] Therefore, how to construct a microbial compound fertilizer with strong salt and alkali tolerance, interspecific synergistic stability and environmental adaptability has become the key to breaking through the bottleneck of microbial treatment of saline-alkali soil. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a kind of microbial compound fertilizer for saline-alkali soil improvement and its preparation method.For Pseudomonas sp., Burkholderia sp., Paenibacillus sp., Bacillus mucilaginosus and Bacillus soil, co-culture in modified LB medium containing sodium chloride, domestication by 10 times of continuous passage, screen out stable combination, expand culture by salt-alkali gradient domestication, obtain compound bacterial solution by ultrasonic treatment and centrifugal concentration, then mix chitosan solution, compound bacterial solution and humic acid mixture to form emulsion, crosslink by calcium chloride and glutaraldehyde to form microcapsules, granulate, pre-dry, dry and spray outer coating to obtain embedded microcapsules, then mix with mature sheep manure, monoammonium phosphate and potassium sulfate to obtain a kind of microbial compound fertilizer for saline-alkali soil improvement.The salt-tolerant bacterial group screened by the method can effectively adapt to salt-alkali stress environment, has high survival rate, can effectively relieve saline-alkali stress, and improve vegetation survival rate and soil quality.

[0006] The present application provides a kind of microbial compound fertilizer for saline-alkali soil improvement and its preparation method, including the following steps: selecting Pseudomonas sp., Burkholderia sp., Paenibacillus sp., Bacillus mucilaginosus and Bacillus soil, co-culture in medium containing sodium chloride, and screening out stable combination.

[0007] Expand culture of the screened stable combination by salt-alkali gradient domestication process, and obtain compound bacterial solution by ultrasonic treatment.

[0008] Stir chitosan solution, compound bacterial solution and humic acid mixture uniformly to form emulsion, crosslink the emulsion by calcium chloride solution and glutaraldehyde solution to prepare microcapsules.

[0009] Put microcapsules into a granulator, pre-dry, dry and spray outer coating to obtain embedded microcapsules, then mix embedded microcapsules with mature sheep manure, monoammonium phosphate, potassium sulfate and straw powder to obtain microbial compound fertilizer.

[0010] Preferably, the co-culture medium is a modified LB medium, which comprises peptone 10 g / L, yeast extract 5 g / L, sodium chloride 30 g / L, soluble starch 5 g / L, potassium feldspar powder 2 g / L, pH = 8.5-9.0, and the medium is treated by 121℃ sterilization for 20 min.

[0011] Preferably, the co-culture is adaptive domestication culture by continuous passage for 10 times, and the strain ratio is adjusted to OD600 difference ≤0.1 and interspecies antagonism rate ≤5% for each generation.

[0012] Preferably, the salt and alkali gradient domestication process is as follows: the initial salinity is 1% sodium chloride by mass fraction, the salinity gradient is increased by 0.3%-0.5% for each passage, the final salinity reaches 3%-5%, and the alkalinity is gradually increased from pH = 7.5 to pH = 8.5-9.0.

[0013] Preferably, the power of the ultrasonic treatment is 20-30 W, the pulse mode is adopted, each treatment is 5-10 min, the interval is 4 hours, the culture time is 72-96 hours, the compound bacterial liquid is concentrated by centrifugation, the centrifugal speed is 5000-8000 r / min, the centrifugation time is 5-10 min, and the concentration of the bacterial liquid after concentration is 10 9 CFU / mL.

[0014] Preferably, the chitosan solution is low molecular weight chitosan dissolved in 1-3% glacial acetic acid, the mass ratio of low molecular weight chitosan to glacial acetic acid is 1: (6-8), and the mass fraction of the humic acid mixed solution is 5-10%, the mass ratio of the chitosan solution, the compound bacterial liquid and the humic acid mixed solution is 1: (4-5): (8-9).

[0015] Preferably, the concentration of the calcium chloride solution is 0.3-0.5 mol / L, the volume ratio of the CaCl2 solution to the emulsion is 1: (4-6), the emulsion and the CaCl2 solution are mixed and stirred to form primary cross-linked microcapsules, and the concentration of the glutaraldehyde solution is 0.1-0.5%, the volume ratio of the glutaraldehyde solution to the primary cross-linked microcapsules is 1: (40-50).

[0016] Preferably, the granulator is a disc granulator, the rotating speed is 25 r / min, the inclination angle is 48°, 2% chitosan solution by mass fraction is added during granulation, the pre-drying is cold air pre-drying at 30-40℃ for 1-2 h, the drying temperature is 45-50℃, the drying time is 1.5-3 hours, and the solution for spraying the outer coating is a nano-silicon dioxide suspension with a mass fraction of 0.5-0.8%.

[0017] Preferably, the mass ratio of the embedded microcapsules, decomposed sheep manure, monoammonium phosphate, potassium sulfate and straw powder is (30-40):(10-20):(15-20):(5-10):(10-25).

[0018] The application also provides a microbial compound fertilizer for improving saline-alkali soil treatment.

[0019] Beneficial technical effects:

[0020] The screened stable flora combination is expanded by using a salt-alkali gradient acclimation process, and finally a flora with genetic stability and true high salt-alkali resistance is obtained. The flora acclimated by the salt-alkali gradient can be slightly disturbed by low-intensity, pulse-type ultrasonic waves, slight mechanical vibration and cavitation effect, thereby enhancing the permeability of the cell membrane, promoting the absorption of nutrients and the discharge of metabolic waste, and thus accelerating the growth and reproduction of the microorganisms. The gradient acclimation optimizes the metabolic pathways to adapt to the high-salt-alkali environment nutrition mode, and the ultrasonic waves enhance the carbon and nitrogen source absorption efficiency by improving the cell membrane fluidity, thereby improving the material conversion and biomass output efficiency. Through genetic adaptability modification of the microorganisms by the salt-alkali gradient acclimation and physiological activity excitation by the ultrasonic treatment, the comprehensive stress resistance, enzyme activity, metabolic activity and flora cooperation efficiency of the functional flora are significantly improved by the double means, so that the compound microbial agent can survive and work efficiently after being applied to the harsh saline-alkali soil environment, can effectively relieve the stress of the saline-alkali soil, and improve the vegetation survival rate and soil quality. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a process flow chart of the preparation method of the microbial compound fertilizer for improving saline-alkali soil treatment. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and beneficial effects of the present application clearer, the present application will be further described in detail below in conjunction with the embodiments. However, it should not be understood as limiting the scope of the present application to the following embodiments. Without departing from the above method idea of the present application, all other embodiments obtained by those skilled in the art without making creative efforts shall fall within the scope of protection of the present application.

[0023] In the present application, the terms used in the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application.

[0024] In the present application, the singular forms "is", "a", "any" and "the" used in the present application are intended to include the plural forms, unless the context clearly indicates otherwise.

[0025] Furthermore, the terms "first" and "second" appearing in this application are for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0026] The following will describe in detail, with reference to different embodiments, a microbial compound fertilizer for the treatment and improvement of saline-alkali land and its preparation method provided in this application.

[0027] *Pseudomonas sp.*, accession number MCCC1A00089, China Marine Microbial Culture Collection Center; *Burkholderia sp.*, accession number CGMCCNo.16428, China General Microbial Culture Collection Center; *Paenibacillus sp.*, accession number CCTCCAB2022354, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences; *Bacillus mucilaginosus*, accession number CICC21700, Gansu Academy of Sciences Institute of Biology; *Bacillus soil*, accession number CGMCC1.8813, China General Microbial Culture Collection Center. Bacillus subtilis, accession number CICC 10073, China Industrial Microbiological Culture Collection Center; Bacillus megaterium, accession number CGMCC No. 15070, China General Microbiological Culture Collection Center; Bacillus thuringiensis, accession number CGMCC No. 27453, China General Microbiological Culture Collection Center; Bacillus licheniformis, accession number CGMCC NO. 14106, China General Microbiological Culture Collection Center; Bacillus amyloliquefaciens, accession number CICC 24192, China Industrial Microbiological Culture Collection Center; Pseudomonas fluorescens, accession number CCTCC M2012260, China Center for Type Culture Collection.

[0028] Example 1:

[0029] like Figure 1 As shown, a method for preparing a microbial compound fertilizer for the treatment and improvement of saline-alkali land includes the following steps:

[0030] 1. Screening and propagation of functional strains

[0031] Strain screening: Pseudomonas, Burkholderia, Bacillus subtilis, Bacillus mucilaginosus, and Bacillus spp. were co-cultured for acclimatization (initially inoculated at a bacterial volume ratio of 1:1:1:1:1, with a concentration of 10 for each bacterial culture). 8 The strains (cfu / mL) were passaged 10 times in modified LB medium, with the OD600 difference adjusted to ≤0.1 per generation. Stable combinations with interspecies antagonism ≤5% were screened. The modified LB medium consisted of 10 g / L peptone, 5 g / L yeast extract, 30 g / L sodium chloride, 5 g / L soluble starch, and 2 g / L potassium feldspar powder, with a pH of 8.5. The medium was sterilized at 121℃ for 20 min. After screening, the bacterial count was performed using the plate count method, and the bacterial count ratio of Pseudomonas:Burkholderia:Bacillus:Geloidobacterium:Agrobacterium was 1:1.06:0.99:0.98:1.01.

[0032] During fermentation, the initial salinity was 1% NaCl (by mass), increasing by 0.4% with each subculture, eventually reaching a NaCl concentration of 4%. The alkalinity gradient gradually increased from pH 7.5 to pH 8.5. Pulsed ultrasonic therapy (on / off = 1s / 5s), 25W, was used for 7 minutes each time, spaced 4 hours apart, for a total of 90 hours. The bacterial culture was then concentrated to 10 μL by centrifugation (6000 rpm, 5 minutes). 9 The concentration of cfu / mL was used to obtain a compound bacterial solution.

[0033] 2. Encapsulation of chitosan functional components

[0034] Low molecular weight chitosan was dissolved in a 2% glacial acetic acid solution. A mixture of compound bacterial solution and 8% humic acid was added, and the mixture was stirred until homogeneous. The mixture was then transferred to a high-speed emulsifier and sheared at 2000 rpm for 10 minutes to form an emulsion. A 0.4 mol / L CaCl2 solution was added dropwise, and the mixture was stirred at 35°C for 10 minutes to form primary cross-linked microcapsules. Then, a 0.3% glutaraldehyde solution was added, and the mixture was stirred for another 10 minutes to complete the double cross-linking, thus preparing the microcapsules. The mass ratio of low molecular weight chitosan to glacial acetic acid was 1:7, the volume ratio of CaCl2 solution to emulsion was 1:5, the mass ratio of chitosan solution, compound bacterial solution, and humic acid mixture was 1:4.5:8.5, and the volume ratio of glutaraldehyde solution to primary cross-linked microcapsules was 1:45.

[0035] 3. Compound fertilizer molding

[0036] Microcapsules were fed into a disc granulator (25 r / min, 48° inclination), and 2% chitosan solution was added. The granulation particle size was controlled at 3-5 mm. After granulation, the microcapsules were pre-dried with cold air at 35°C for 1.5 h, then transferred to a fluidized bed dryer and dried at 45°C for 2 h. Finally, a 0.6% (w / w) nano-SiO2 suspension was sprayed as an outer coating to obtain encapsulated microcapsules. The encapsulated microcapsules, well-rotted sheep manure, monoammonium phosphate, potassium sulfate, and straw powder were mixed to obtain a microbial compound fertilizer. The mass ratio of encapsulated microcapsules, well-rotted sheep manure, monoammonium phosphate, potassium sulfate, and straw powder was 35:15:18:7:20.

[0037] Example 2:

[0038] like Figure 1 As shown, a method for preparing a microbial compound fertilizer for the treatment and improvement of saline-alkali land includes the following steps:

[0039] 1. Screening and propagation of functional strains

[0040] Strain screening: Pseudomonas, Burkholderia, Bacillus subtilis, Bacillus mucilaginosus, and Bacillus spp. were co-cultured for acclimatization (initially inoculated at a bacterial volume ratio of 1:1:1:1:1, with a concentration of 10 for each bacterial culture). 8 The strains (cfu / mL) were passaged 10 times in modified LB medium, with the OD600 difference adjusted to ≤0.1 per generation. Stable combinations with interspecies antagonism ≤5% were screened. The modified LB medium consisted of 10 g / L peptone, 5 g / L yeast extract, 30 g / L sodium chloride, 5 g / L soluble starch, and 2 g / L potassium feldspar powder, with a pH of 8.7. The medium was sterilized at 121℃ for 20 min. After screening, the bacterial count was performed using the plate count method, and the bacterial count ratio of Pseudomonas:Burkholderia:Bacillus-like bacteria:Bacillus mucilaginosus:Bacillus stolonifera was 1:1.06:0.99:0.98:1.01.

[0041] During fermentation, the initial salinity typically starts at 1% NaCl, increasing by 0.3% with each subculture until a final concentration of 3% is reached. The alkalinity gradient gradually increases from pH 7.5 to pH 8.5. Ultrasonic power is 20W, pulse mode (on / off = 1s / 5s), 5 minutes per pulse, spaced 4 hours apart, for a total culture time of 72 hours. The culture is then concentrated to 10 μL by centrifugation (5000 rpm, 10 minutes). 9 The concentration of cfu / mL was used to obtain a compound bacterial solution.

[0042] 2. Encapsulation of chitosan functional components

[0043] Low molecular weight chitosan was dissolved in a 1% glacial acetic acid solution. A mixture of compound bacterial solution and 5% humic acid (volume ratio 1:2) was added, and the mixture was stirred until homogeneous. The mixture was then transferred to a high-speed emulsifier and sheared at 2000-3000 rpm for 10-15 minutes to form an emulsion. A 0.3 mol / L CaCl2 solution was added dropwise (CaCl2 solution volume was 1 / 5 of the emulsion volume), and the mixture was stirred at 30-40℃ for 10-20 minutes to form primary cross-linked microcapsules. Then, a 0.1% glutaraldehyde solution was added (glutaraldehyde solution volume was 1 / 50 of the primary cross-linked microcapsule volume), and stirring was continued for 10-15 minutes to complete the double cross-linking, thus preparing the microcapsules. The volume ratio of chitosan solution to the compound bacterial solution-humic acid mixture was 1:4. The mass ratio of low molecular weight chitosan to glacial acetic acid is 1:6, the volume ratio of CaCl2 solution to emulsion is 1:5, the mass ratio of chitosan solution, composite bacterial solution and humic acid mixture is 1:4:8, and the volume ratio of glutaraldehyde solution to primary cross-linked microcapsules is 1:40.

[0044] 3. Compound fertilizer molding

[0045] Microcapsules were fed into a disc granulator (25 r / min, 48° inclination), and 2% chitosan solution was added. The granulation particle size was controlled at 3-5 mm. After granulation, the microcapsules were pre-dried with cold air at 30°C for 1 hour, then transferred to a fluidized bed dryer at 45°C for 1.5 hours. Finally, a 0.5% nano-SiO2 suspension was sprayed as an outer coating to obtain encapsulated microcapsules. The encapsulated microcapsules, well-rotted sheep manure, monoammonium phosphate, potassium sulfate, and straw powder were mixed to obtain a microbial compound fertilizer. The mass ratio of microcapsules, well-rotted sheep manure, monoammonium phosphate, potassium sulfate, and straw powder was 30:10:15:5:10.

[0046] Example 3:

[0047] like Figure 1 As shown, a method for preparing a microbial compound fertilizer for the treatment and improvement of saline-alkali land includes the following steps:

[0048] 1. Screening and propagation of functional strains

[0049] Strain screening: Pseudomonas, Burkholderia, Bacillus subtilis, Bacillus mucilaginosus, and Bacillus spp. were co-cultured for acclimatization (initially inoculated at a bacterial volume ratio of 1:1:1:1:1, with a concentration of 10 for each bacterial culture). 8The strains (cfu / mL) were passaged 10 times in modified LB medium, with the OD600 difference adjusted to ≤0.1 per generation. Stable combinations with interspecies antagonism ≤5% were screened. The modified LB medium consisted of 10 g / L peptone, 5 g / L yeast extract, 30 g / L sodium chloride, 5 g / L soluble starch, and 2 g / L potassium feldspar powder, with a pH of 9.0. The medium was sterilized at 121℃ for 20 min. After screening, the bacterial count was performed using the plate count method, and the bacterial count ratio of Pseudomonas:Burkholderia:Bacillus-like bacteria:Bacillus mucilaginosus:Bacillus stolonifera was 1:1.06:0.99:0.98:1.01.

[0050] During fermentation, the initial salinity is typically started at 1% NaCl, increasing by 0.5% with each subculture until a final concentration of 5% is reached. The alkalinity gradient gradually increases from pH 7.5 to pH 9.0. Ultrasonic power is 30W, pulse mode (on / off = 1s / 5s), each session lasting 10 minutes, spaced 4 hours apart, for a total cultivation period of 96 hours. The bacterial culture is then concentrated to 10 μL by centrifugation (8000 rpm, 5 minutes). 9 The concentration of cfu / mL was used to obtain a compound bacterial solution.

[0051] 2. Encapsulation of chitosan functional components

[0052] Low molecular weight chitosan was dissolved in 3% glacial acetic acid solution, and a mixture of compound bacterial solution and 10% humic acid was added. After stirring evenly, the mixture was transferred to a high-speed emulsifier and sheared at 3000 r / min for 15 min to form an emulsion. 0.5 mol / L CaCl2 solution was added dropwise, and the mixture was stirred at 40℃ for 20 min to form primary cross-linked microcapsules. Then, 0.5% glutaraldehyde solution was added, and stirring was continued for 15 min to complete double cross-linking, thus preparing the microcapsules. The mass ratio of low molecular weight chitosan to glacial acetic acid was 1:8, the volume ratio of CaCl2 solution to emulsion was 1:5, the mass ratio of chitosan solution, compound bacterial solution, and humic acid mixture was 1:4.5:8.5, and the volume ratio of glutaraldehyde solution to primary cross-linked microcapsules was 1:45.

[0053] 3. Compound fertilizer molding

[0054] Microcapsules were fed into a disc granulator (25 r / min, 48° inclination), and 2% chitosan solution was added. The granulation particle size was controlled at 3-5 mm. After granulation, the microcapsules were pre-dried with cold air at 40°C for 2 hours, then transferred to a fluidized bed dryer and dried at 50°C for 3 hours. Finally, a 0.8% nano-SiO2 suspension was sprayed as an outer coating to obtain encapsulated microcapsules. The encapsulated microcapsules, well-rotted sheep manure, monoammonium phosphate, potassium sulfate, and straw powder were mixed to obtain a microbial compound fertilizer. The mass ratio of encapsulated microcapsules, well-rotted sheep manure, monoammonium phosphate, potassium sulfate, and straw powder was 40:20:20:10:25.

[0055] Comparative Example 1:

[0056] A method for preparing a microbial compound fertilizer for the treatment and improvement of saline-alkali land includes the following steps:

[0057] 1. Screening and propagation of functional strains

[0058] Strains were screened using Bacillus subtilis, Bacillus megaterium, and Bacillus thuringiensis (inoculated at a volume ratio of 1:1:1, with each bacterial culture having a concentration of 10 μL). 8 The cfu / mL of the sample was inoculated into ordinary LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0, sterilized at 121℃ for 20 min) and directly mixed and cultured.

[0059] During fermentation, a fixed salinity of 3% NaCl was used, and the pH was maintained at 7.0. The culture was then statically incubated for 72 hours, followed by centrifugation at 6000 rpm for 10 minutes to concentrate the bacterial solution to 10⁻⁶. 9 The concentration of cfu / mL was used to obtain a compound bacterial solution.

[0060] 2. Sodium alginate encapsulation treatment

[0061] Dissolve medium molecular weight sodium alginate in deionized water at a mass ratio of 1:10. Add a mixture of compound bacterial solution and 5% potassium humate (volume ratio of compound bacterial solution to potassium humate 1:3). Stir evenly and then stir with a magnetic stirrer at 1000 r / min for 20 min to form a mixture. Add 0.3 mol / L calcium chloride solution (calcium chloride solution volume accounts for 1 / 4 of the mixture volume). Stir at 25℃ for 30 min to form microcapsules. The volume ratio of sodium alginate solution to compound bacterial solution-potassium humate mixture is 1:3.

[0062] 3. Compound fertilizer molding

[0063] Microcapsules were fed into a rotary drum granulator (30 r / min, 40° inclination), and 3% starch solution was added as a binder. The granulation particle size was controlled at 3 mm. After granulation, the microcapsules were directly dried in a 60°C oven. The dried granules were then mixed with 15% well-rotted cow manure, 10% urea, 20% superphosphate, and 25% rice husk powder to obtain a microbial compound fertilizer. The mass ratio of the dried granules to well-rotted cow manure, urea, superphosphate, and rice husk powder was 30:15:10:20:25.

[0064] Comparative Example 2:

[0065] A method for preparing a microbial compound fertilizer for the treatment and improvement of saline-alkali land includes the following steps:

[0066] 1. Screening and propagation of functional strains

[0067] Strains were selected for bacterial strain screening: Bacillus licheniformis, Bacillus amyloliquefaciens, and Pseudomonas fluorescens were used (initially inoculated at a bacterial volume ratio of 2:1:1, with a concentration of 10 for each bacterial strain). 8 (cfu / mL) was inoculated into nutrient broth medium (beef extract 3 g / L, peptone 10 g / L, sodium chloride 5 g / L, pH 7.2, sterilized at 121℃ for 15 min), and directly statically cultured without salinity and alkalinity gradient acclimatization.

[0068] Fermentation was carried out in shake flasks at 30°C with shaking at 180 rpm for 48 hours. The bacterial culture was then concentrated to 10 μL by centrifugation at 5000 rpm for 8 minutes. 9 The concentration of cfu / mL was used to obtain a compound bacterial solution.

[0069] 2. Carboxymethyl cellulose encapsulation treatment

[0070] Sodium carboxymethyl cellulose was dissolved in 0.5% dilute hydrochloric acid solution at a mass ratio of 1:6. A mixture of compound bacterial solution and 8% lignin (volume ratio of compound bacterial solution to lignin 1:1) was added, and the mixture was stirred until homogeneous. The mixture was then transferred to a colloid mill and treated at 1500 rpm for 8 minutes to form a suspension. A 0.3 mol / L alum solution was added dropwise (the volume of alum solution was 1 / 4 of the suspension volume), and the mixture was stirred at 25°C for 25 minutes to form microcapsules. The volume ratio of carboxymethyl cellulose solution to the compound bacterial solution-lignin mixture was 1:3.

[0071] 3. Compound fertilizer molding

[0072] Microcapsules were fed into an extrusion granulator (4mm aperture), and a 1% polyvinyl alcohol solution was added as a binder. After granulation, the mixture was first dried at 35℃ for 3 hours, then dried at 55℃, and finally sprayed with a 1% paraffin emulsion as an outer coating. The treated microcapsules, well-rotted chicken manure, ammonium nitrate, diammonium phosphate, potassium chloride, and peanut shell powder were mixed to obtain a microbial compound fertilizer. The mass ratio of microcapsules, well-rotted chicken manure, ammonium nitrate, diammonium phosphate, potassium chloride, and peanut shell powder was 30:15:12:12:8:22.

[0073] Viable bacteria count was performed using the dilution plate count method on modified LB medium. Total nutrients (N+P2O5+K2O) included the sum of total nitrogen, available phosphorus, and total potassium. Total nitrogen was determined by distillation titration. The microbial compound fertilizer was digested with sulfuric acid to convert various forms of nitrogen into ammonium nitrogen. Ammonia was released by alkali distillation, absorbed with boric acid solution, and titrated with a standard sulfuric acid solution using methyl red-methylene blue as an indicator. The total nitrogen content was calculated based on the volume of standard solution consumed. Available phosphorus was extracted using an extractant. The phosphorus in the extract reacted with ammonium vanadomolybdate to form a yellow complex. The absorbance was measured at 420 nm, and the available phosphorus content was calculated using a standard curve. Total potassium content was determined by sulfuric acid-hydrogen peroxide digestion of the microbial compound fertilizer to convert potassium into soluble potassium ions. The potassium content was determined by flame photometry and calculated by comparing with a standard solution.

[0074] For the contamination rate, dissolve 10g of microbial compound fertilizer in 90mL of sterile diluent to prepare a 1:10 dilution. Then, serially dilute to the appropriate concentration, selecting 2-3 dilutions. Spread 0.1mL of each dilution onto nutrient agar and contamination-selective medium, performing three replicates for each. Incubate at 37℃. Moisture content is tested according to GB / T8576-2010 "Determination of Free Water Content in Fertilizers - Vacuum Oven Method". Organic matter content is commonly tested using the potassium dichromate oxidation-external heating method. pH is tested according to GB / T17767.1-2008 "Determination of Organic-Inorganic Compound Fertilizers - Part 1: Determination of pH Value".

[0075] Table 1. Test results of the microbial compound fertilizers prepared in the examples and comparative examples.

[0076] Viable bacterial count (cfu, million / g) Total nutrient (%) Contaminant rate (%) Moisture (%) Organic matter (%) pH Example 1 0.84 22 12 15 42 7.2 Example 2 0.86 23 14 13 44 7.3 Example 3 0.81 21 13 12 43 7.5 Comparative Example 1 0.46 20 25 16 31 7.8 Comparative Example 2 0.68 17 23 18 33 7.6

[0077] Diluted microbial compound fertilizer solution was inoculated into lactose bile salt fermentation tubes and cultured at 36±1℃ for 24-48 hours. Acid and gas production were observed. Positive tubes were transferred to EC broth tubes and cultured at 44.5±0.5℃ for 48 hours. Gas production was counted, and the results were expressed as "cells / g". The mortality rate of Ascaris eggs was determined by separating Ascaris eggs from the compound fertilizer using a water washing and sedimentation method, and observing egg morphology under a microscope. The eggs were then cultured in a 37℃ medium, and the ratio of live eggs (active embryos) to dead eggs (withered embryos) was counted. The mortality rate was calculated as (number of dead eggs / total number of eggs) × 100%. Heavy metal content was tested after digestion of the compound fertilizer with nitric acid and perchloric acid. Arsenic and mercury were determined using atomic fluorescence spectrometry (AFS); chromium and lead were determined using flame atomic absorption spectrophotometry (FAAS). Results were expressed as "mg / kg".

[0078] Table 2. Results of the harmlessness test of the microbial compound fertilizers prepared in the examples and comparative examples.

[0079] Fecal coliform (number / g) Ascarid egg mortality rate (%) Arsenic (mg / kg) Chromium (mg / kg) Lead (mg / kg) Mercury (mg / kg) Example 1 10 98 Not detected Not detected Not detected Not detected Example 2 10 97 Not detected Not detected Not detected Not detected Example 3 10 98 Not detected Not detected Not detected Not detected Comparative Example 1 64 85 7.0 15.0 6.0 0.5 Comparative Example 2 58 90 5.0 12.0 4.0 0.4

[0080] As shown in Table 1, the results of viable bacterial count, total nutrients, contamination rate, and organic matter in Examples 1-3 were superior to those in Comparative Examples 1-2. This is because Examples 1-3 employed a "co-culture adaptive acclimatization" method, adjusting the ratio through 10 subcultures until the OD600 difference was ≤0.1, ultimately selecting a stable combination with an interspecific antagonism rate ≤5%. In the fertilizer microenvironment, functional bacteria rapidly multiply to occupy dominant ecological niches, secreting antibacterial substances to inhibit the growth of contaminating bacteria. This acclimatization process eliminated competitive inhibition between strains, allowing functional bacteria such as Pseudomonas to coexist stably and avoiding viable bacterial loss caused by "interspecific mutual exclusion." In contrast, Comparative Example 1 directly mixed three Bacillus species, and Comparative Example 2 used Bacillus licheniformis and other strains without co-acclimatization, resulting in intense interspecific competition and the death of some strains, leading to a decrease in the total viable bacterial count. The fermenter cultures in Examples 1-3 employed a salinity / alkalinity gradient acclimatization method, using stepwise stress to induce the strains to synthesize salt-tolerance-related proteins and alkali-tolerance enzyme systems, significantly enhancing their survival ability in the high-salt, high-pH environment of saline-alkali land. Ultrasonic treatment was introduced during cultivation to promote cell membrane permeability through mechanical vibration, accelerating nutrient absorption and metabolism, and improving reproductive efficiency. The combination of salinity / alkalinity gradient acclimatization and ultrasonic treatment significantly enhanced the stress resistance, metabolic activity, interspecific compatibility, and environmental adaptability of the composite strains. In contrast, Comparative Example 1, with a fixed salinity of 3% and pH of 7.0, did not adapt to the high salinity / alkalinity environment, while Comparative Example 2, without salinity / alkalinity acclimatization, was prone to death due to osmotic pressure imbalance or enzyme inactivation in the saline-alkali environment, resulting in a low viable cell survival rate. Examples 1-3 used monoammonium phosphate and potassium sulfate. The phosphorus in monoammonium phosphate was in the form of water-soluble PO4. 3- It exists and is easily absorbed by crops; potassium sulfate contains potassium ions (K+). + It can replace Na in the soil + It has the dual function of fertilization and improvement of saline-alkali land. Comparative Example 1 used superphosphate and urea, and Comparative Example 2 used ammonium nitrate and potassium chloride. The nutrient utilization rate of both examples was lower than that of Examples 1-3.

[0081] As shown in Table 2, the fecal coliform, Ascaris egg mortality, and heavy metal content test results in Examples 1-3 were all superior to those in Comparative Examples 1-2. This is because the Pseudomonas bacteria screened in Examples 1-3 are natural producers of antibacterial agents, and Pseudomonas bacteria can produce phenazine compounds, which have a direct inhibitory effect on the growth of fecal coliforms; while Bacillus subtilis in Comparative Example 1 and Bacillus licheniformis in Comparative Example 2 have a narrower antibacterial spectrum and weaker antagonistic ability against intestinal pathogens. In Examples 1-3, humic acid was added during the encapsulation process. The carboxyl and phenolic hydroxyl groups of humic acid can destroy the chitinous structure of the Ascaris eggshell, increasing its permeability and causing leakage of contents and death; at the same time, humic acid can lower the environmental pH and inhibit the embryonic development of Ascaris eggs. In contrast, potassium humate in Comparative Example 1 and lignin in Comparative Example 2 showed significantly weaker killing ability against Ascaris eggs. In Examples 1-3, *Pseudomonas* can chelate lead and mercury with extracellular polymers, converting them into insoluble sulfides; the organic acids produced by *Bacillus mucilaginosus* can form stable chelates with arsenic and chromium, reducing their bioavailability; and *Burkholderia* can reduce hexavalent chromium to trivalent chromium through respiration. However, the strains in Comparative Examples 1 and 2, which had not undergone heavy metal stress acclimatization, lacked this transformation ability.

[0082] The above results demonstrate and describe the basic principles and main features of this application, as well as its advantages.

[0083] Those skilled in the art should understand that this application is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of this application. Various changes and modifications can be made to this application without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of this application as claimed. The scope of protection of this application is defined by the equivalents of the appended claims.

Claims

1. A method for preparing a microbial compound fertilizer for the treatment and improvement of saline-alkali land, characterized in that, Includes the following steps: Pseudomonas sp., Burkholderia sp., Paenibacillus sp., Bacillus mucilaginosus, and Bacillus soil were selected and co-cultured in a medium containing sodium chloride to screen out stable combinations. The selected stable combinations were cultured using a salt-alkali gradient acclimatization process, and then combined with ultrasonic treatment to obtain a composite bacterial solution. Chitosan solution, compound bacterial solution and humic acid mixture were stirred evenly to form an emulsion. The emulsion was then cross-linked with calcium chloride solution and glutaraldehyde solution to prepare microcapsules. Microcapsules are fed into a granulator, pre-dried, and then coated with an outer coating to obtain encapsulated microcapsules. The encapsulated microcapsules are then mixed with well-rotted sheep manure, monoammonium phosphate, potassium sulfate, and straw powder to obtain microbial compound fertilizer.

2. The method for preparing a microbial compound fertilizer for saline-alkali land improvement according to claim 1, characterized in that, The culture medium used for co-culture is a modified LB medium, which includes 10 g / L peptone, 5 g / L yeast extract, 30 g / L sodium chloride, 5 g / L soluble starch, and 2 g / L potassium feldspar powder, with a pH of 8.5-9.

0. The medium is sterilized at 121°C for 20 min.

3. The method for preparing a microbial compound fertilizer for saline-alkali land improvement according to claim 1, characterized in that, The co-culture is an adaptive domestication culture that is continuously passaged 10 times. The proportion of strains is adjusted in each generation until the difference in OD600 is ≤0.1 and the interspecific antagonism rate is ≤5%.

4. The method for preparing a microbial compound fertilizer for saline-alkali land improvement according to claim 1, characterized in that, The salt-alkali gradient acclimatization process is as follows: the initial salinity is 1% sodium chloride by mass, and the salinity is increased by 0.3%-0.5% with each generation, eventually reaching 3%-5% salinity, and the alkalinity is gradually increased from pH=7.5 to pH=8.5-9.

0.

5. The method for preparing a microbial compound fertilizer for saline-alkali land improvement according to claim 1, characterized in that, The ultrasonic treatment power is 20-30W, using pulse mode, with each treatment lasting 5-10 minutes, repeated every 4 hours, and the culture time is 72-96 hours. The composite bacterial solution is concentrated by centrifugation at a speed of 5000-8000 r / min for 5-10 minutes, resulting in a bacterial solution concentration of 10%. 9 CFU / mL.

6. The method for preparing a microbial compound fertilizer for saline-alkali land improvement according to claim 1, characterized in that, The chitosan solution is low molecular weight chitosan dissolved in 1-3% glacial acetic acid, with a mass ratio of low molecular weight chitosan to glacial acetic acid of 1:(6-8). The humic acid mixture has a mass fraction of 5-10%, and the mass ratio of chitosan solution, compound bacterial solution and humic acid mixture is 1:(4-5):(8-9).

7. The method for preparing a microbial compound fertilizer for saline-alkali land improvement according to claim 1, characterized in that, The concentration of the calcium chloride solution is 0.3-0.5 mol / L, the volume ratio of CaCl2 solution to emulsion is 1:(4-6), the emulsion and CaCl2 solution are mixed and stirred to form primary cross-linked microcapsules, the concentration of the glutaraldehyde solution is 0.1-0.5%, and the volume ratio of glutaraldehyde solution to primary cross-linked microcapsules is 1:(40-50).

8. The method for preparing a microbial compound fertilizer for saline-alkali land improvement according to claim 1, characterized in that, The granulator is a disc granulator with a rotation speed of 25 r / min and an inclination angle of 48°. During granulation, a chitosan solution with a mass fraction of 2% is added. The pre-drying is carried out by cold air pre-drying at 30-40°C for 1-2 hours. The drying temperature is 45-50°C and the drying time is 1.5-3 hours. The solution for spraying the outer coating is a nano-silica suspension with a mass fraction of 0.5-0.8%.

9. A method for preparing a microbial compound fertilizer for saline-alkali land improvement according to claim 1, characterized in that, The mass ratio of the encapsulated microcapsules, decomposed sheep manure, monoammonium phosphate, potassium sulfate and straw powder is (30-40): (10-20): (15-20): (5-10): (10-25).

10. A microbial compound fertilizer for the treatment and improvement of saline-alkali land, characterized in that, The microbial compound fertilizer for saline-alkali land treatment and improvement is prepared by any one of claims 1-9.

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