Alkalibacter sp., isolation and screening method and application thereof in acid soil improvement

Abstract

The application discloses an alkali-producing bacterium, a separation and screening method and application of the alkali-producing bacterium in acid soil improvement, and belongs to the technical field of microorganisms. The alkali-producing bacterium is Providencia rettgeri, and the Latin name is Providencia rettgeri. The Providencia rettgeri is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 38612, and the preservation date is May 9, 2026. The strain is a gram-positive bacillus, and has a significant alkali-producing capacity in an acid environment. The strain is obtained from alkaline soil through gradient dilution coating and acid environment rescreening, and soil potting experiments prove that the strain can effectively improve the pH value of acid soil, reduce the content of exchangeable aluminum, and relieve the harm of aluminum toxicity. The alkali-producing bacterium provided by the application is environment-friendly, has stable improvement effect, and has a wide application prospect in the field of acid soil improvement.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to an alkali-producing bacterium, a method for its isolation and screening, and its application in the improvement of acidic soil. Background Technology

[0002] Soil acidification is one of the most serious soil degradation problems globally, posing a significant threat to sustainable agricultural development and ecological security. The harmful effects of acidic soil on crop growth are multifaceted, cumulative, and irreversible. First, excessively low soil pH directly affects the morphogenesis and physiological functions of crop roots. Studies show that when soil pH is below 5.0, root growth in most crops is significantly inhibited, manifesting as short, sparse, and browned roots, root tip necrosis, and a sharp decline in the ability to absorb water and nutrients. Second, the large-scale activation of aluminum ions in acidic soil is the primary cause of inhibited crop growth. Under acidic conditions, aluminosilicate minerals in the soil dissolve, releasing large amounts of exchangeable aluminum ions. These aluminum ions bind to phospholipids and proteins on the cell membranes of crop roots, damaging the cell membrane structure and function, leading to the leakage of intracellular electrolytes. Simultaneously, aluminum ions inhibit root cell division and elongation, interfere with intracellular calcium signaling, and affect the absorption of water and nutrients by the roots. Severe aluminum toxicity can lead to complete root necrosis, plant wilting, and death. Third, the availability of nutrients in acidic soils is significantly reduced. Under acidic conditions, phosphorus in the soil combines with iron and aluminum ions to form insoluble iron phosphate and aluminum phosphate precipitates, which are difficult for crops to absorb and utilize. Even with large applications of phosphate fertilizer, the utilization rate is less than 10%. Furthermore, the solubility and mobility of micronutrients such as calcium, magnesium, boron, and molybdenum are significantly reduced in acidic soils, leading to nutrient deficiency symptoms in crops. Fourth, the activity of beneficial microorganisms in acidic soils is severely inhibited. Most beneficial microorganisms in the soil, such as nitrifying bacteria, nitrogen-fixing bacteria, and phosphate-solubilizing bacteria, thrive in neutral or slightly alkaline environments. Under acidic conditions, the number and activity of these microorganisms decrease significantly, resulting in slow decomposition of soil organic matter, hindered nitrogen transformation, and reduced soil fertility.

[0003] Currently, acidic soil improvement mainly employs three methods: chemical, physical, and biological. Chemical improvement is the most widely used method, primarily neutralizing soil acidity by applying alkaline substances such as lime, limestone powder, dolomite powder, and gypsum. However, chemical amendments have several insurmountable limitations. First, their effects are short-lived. Chemical amendments can only neutralize the acidity of the soil surface and are difficult to penetrate into deeper soil layers. Moreover, their effects gradually disappear with rainwater leaching and crop absorption, requiring continuous application year after year. Second, they can easily lead to the deterioration of soil physicochemical properties. Long-term, excessive application of lime can cause soil compaction, structural damage, and decreased soil aeration and permeability. Furthermore, excessive lime application can lead to excessively high soil pH, affecting the availability of micronutrients such as iron, manganese, and zinc, resulting in nutrient deficiency symptoms in crops. Third, they are costly. The production, transportation, and application of chemical amendments such as lime require significant manpower, material resources, and financial investment, increasing agricultural production costs. Fourth, they pose environmental pollution risks. Excessive application of chemical amendments can lead to the accumulation of basic ions in the soil, causing soil salinization, and can also pollute groundwater and surface water bodies.

[0004] Physical soil improvement methods mainly include deep plowing, topsoil introduction, sand mixing, and increased application of organic fertilizers. These measures aim to alleviate soil acidification by improving soil structure. Deep plowing breaks up the plow pan, increasing soil aeration and permeability, and promoting the maturation of deeper soil layers. Topsoil introduction involves mixing alkaline or fertile topsoil into acidic soil to neutralize acidity and improve soil fertility. Sand mixing improves the physical properties of heavy, acidic soils and increases soil porosity. Increased application of organic fertilizers increases soil organic matter content, improves soil structure, and enhances soil buffering capacity. However, physical improvement methods also have significant drawbacks. Deep plowing and topsoil introduction involve large-scale engineering projects and are costly, making them difficult to implement on a large scale. Sand mixing only improves the physical properties of the soil and cannot fundamentally solve the problem of soil acidification. While increased application of organic fertilizers can alleviate soil acidification to some extent, it is slow to take effect and requires long-term application to achieve significant results.

[0005] Biological soil improvement is a novel soil amendment technology that has emerged in recent years, primarily utilizing plants, microorganisms, and their metabolic products to improve acidic soils. Compared to chemical and physical methods, biological soil improvement offers advantages such as environmental friendliness, low cost, and long-lasting effects, making it a hot topic and development direction in acidic soil improvement research. Among these, microbial soil improvement has attracted significant attention due to its rapid and remarkable effects. Alkali-producing microorganisms are a class of microorganisms capable of producing alkaline substances through metabolic activities, thereby increasing the pH value of the environment. They are widely distributed in nature and include bacteria, fungi, and actinomycetes. Alkali-producing microorganisms mainly produce alkali through the following pathways: first, decomposing organic matter to produce alkaline substances such as ammonia and amines; second, consuming carbon dioxide through respiration, thus raising the pH value of the environment; third, exchanging hydrogen ions from the soil into cells through ion exchange; and fourth, producing alkaline metabolic products such as carbonates and bicarbonates.

[0006] However, most of the reported alkali-producing microorganisms have the following problems: First, their alkali-producing capacity is limited, and they have poor adaptability to acidic environments. Most alkali-producing bacteria can only produce alkali in neutral or weakly alkaline environments. In acidic environments, their alkali-producing capacity decreases significantly, making it difficult to significantly raise soil pH in a short period of time. Furthermore, they are difficult to colonize and reproduce in acidic soils, and die quickly, failing to exert a sustained improvement effect. Second, the supporting culture medium technology is lagging behind. Commonly used basic culture media such as LB and NA are widely used, with a single nutrient composition and lack of acid tolerance acclimatization and alkali-producing enhancement design. The strains cultured have poor acid tolerance, low alkali-producing efficiency, and short stable alkali-producing cycles. Third, the bacterial agent formulations are simple, and the improvement effect is unstable. Most are liquid bacterial agents or single-carrier solid bacterial agents, lacking stress protection and continuous nutrient supply. The strains die rapidly after being applied to the soil, resulting in a short-lived improvement effect. Under high inoculum levels, bacterial decomposition can even exacerbate the subsequent pH drop and aluminum toxicity rebound. Fourth, their improvement function is singular. Relying solely on bacterial alkali production to neutralize acidity, they cannot simultaneously achieve aluminum toxicity fixation and soil fertilization, making it difficult to meet the comprehensive management needs of acidic soils. Therefore, screening out an alkali-producing bacterium with strong alkali-producing ability, good adaptability to acidic environments, stable improvement effect, and safety and harmlessness, and developing supporting special culture medium and long-acting bacterial agent, is of great theoretical significance and practical value for promoting the development and application of biological improvement technology for acidic soil. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing an alkali-producing bacterium, a method for its isolation and screening, and its application in the improvement of acidic soils. This alkali-producing bacterium has a strong alkali-producing capacity, exhibits good adaptability to acidic environments, effectively increases the pH value of acidic soils, reduces the content of exchangeable aluminum, alleviates aluminum toxicity, and provides stable improvement effects. It is also environmentally friendly and poses no risk of secondary pollution.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] An alkali-producing bacterium, *Providencia rettgeri*, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38612 on May 9, 2026.

[0010] This invention also provides a method for isolating and screening alkali-producing bacteria, comprising the following steps:

[0011] (1) Samples were collected from alkaline soil and 10 samples were prepared. 4 -10 5 Series of diluted suspensions;

[0012] (2) Spread the diluted suspension on NA medium plates and incubate at 28°C for 48 hours. Select single colonies for purification three times.

[0013] (3) The purified single colony was inoculated into LB medium and cultured at 30°C for 48 hours. The pH value of the bacterial solution was measured to screen out potential alkali-producing bacteria that could increase the pH of the medium.

[0014] (4) Potential alkali-producing bacteria were inoculated into a compound liquid culture medium and cultured at 30°C and 200r / min for 48 hours to screen out strains that could significantly increase the pH of the culture medium in an acidic environment.

[0015] Furthermore, the alkaline soil mentioned in step (1) was collected from the saline-alkali area of ​​Dongying, Shandong.

[0016] Further, the composite liquid culture medium in step (3) includes the following raw materials: glucose, sucrose, soluble starch, tryptone, yeast extract powder, soybean meal powder, corn steep liquor powder, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, manganese sulfate, betaine, and sodium chloride.

[0017] Furthermore, culturing the strain described in step (4) in a compound liquid culture medium for 48 hours can increase the pH value of the culture medium by more than 1.8 units.

[0018] This invention also provides the application of alkali-producing bacteria screened by an isolation and screening method in the improvement of acidic soil.

[0019] Furthermore, the inoculation amount of the alkali-producing bacteria is 5%-10% of the soil weight.

[0020] Furthermore, the acidic soil is red soil, lateritic soil, or red soil with a pH value below 5.5.

[0021] The present invention also provides a compound acidic soil conditioner comprising alkali-producing bacteria screened by the above-described separation and screening method and an agriculturally acceptable carrier.

[0022] Furthermore, the compound acidic soil conditioner includes the following raw materials: alkali-producing bacteria powder, peat, vermiculite powder, corn cob powder, wheat bran, corn flour, soybean meal powder, potassium humate, trehalose, skim milk powder, light calcium carbonate, sodium carboxymethyl cellulose, and fulvic acid.

[0023] Compared with the prior art, the present invention has the following technical advantages:

[0024] 1. This invention develops a specialized composite liquid culture medium targeting the growth and metabolic characteristics and alkali-producing pathway of strain D5-N19, overcoming the limitations of conventional LB medium in terms of single nutrient supply and lack of acid tolerance acclimatization. By matching the energy requirements of the strain at different growth stages with a three-tiered carbon source, continuously supplying alkali-producing substrates with a composite nitrogen source, activating key alkali-producing enzyme systems with mineral elements, and enhancing cell acid tolerance protection with betaine, the invention achieves a simultaneous improvement in the strain's growth rate, alkali-producing efficiency, and acid tolerance. Compared to conventional culture systems, this strain exhibits significantly faster adaptation initiation and greatly enhanced growth and proliferation capabilities; its alkali-producing efficiency and duration of action in acidic environments are significantly optimized; and its survival ability under acid stress is greatly improved, providing a high-quality strain resource with excellent performance for soil improvement applications.

[0025] 2. This invention constructs a triple improvement mechanism of "biological alkali production + chemical buffering + aluminum chelation," overcoming the limitations of single improvement methods. Strain D5-N19 continuously produces alkali through multiple pathways such as ammoniation, respiration, and ion exchange, neutralizing soil hydrogen ions at the source; light calcium carbonate slowly releases carbonate ions, rapidly buffering the localized strong acid environment; potassium humate and fulvic acid fix aluminum ions through functional group complexation, reducing the bioavailability of aluminum. The synergistic effect of these three mechanisms avoids the problems of short-lived effects and easy compaction associated with chemical amendments, while also alleviating the shortcomings of slow-acting and insufficient aluminum toxicity removal associated with single microbial amendments. After 60 days of application, the soil exchangeable aluminum reduction rate reached over 75%, and the pH value steadily increased by more than 0.8 units without rebounding, achieving simultaneous and long-term control of acidity improvement and aluminum toxicity mitigation.

[0026] 3. The composite amendment of this invention constructs a three-dimensional colonization protection system of "carrier protection - nutrient supply - buffer protection," overcoming the technical bottlenecks of low survival rate and short survival period of strains in conventional inoculants. The porous composite carrier provides microecological protection for the strains, avoiding damage from strong acids, aluminum toxicity, and protozoa; the gradient nutrient system provides continuous nutrients throughout the entire cycle of strain recovery, colonization, and alkali production; the triple protection mechanism significantly enhances the strains' stress resistance. Compared with single liquid inoculants, the 7-day survival rate of the strains in acidic soil is significantly improved, and the survival period is extended from 15 days to over 60 days, with the number of viable bacteria in the soil remaining at 6.82 × 10⁻⁶ after 60 days. 5CFU / g or higher. This formula fundamentally alleviates the common problem of alkali-producing bacteria struggling to colonize and rapidly dying in acidic soils, providing core technological support for the long-term application of microbial amendments.

[0027] 4. This invention, through the combination of continuously colonizing functional strains and slow-release amendment components, alleviates the shortcomings of existing amendment technologies, such as short-lived effects and easy rebound. The strains can form stable microbial communities in the soil, continuously producing alkali to maintain soil pH; the carrier and nutrient components degrade slowly, continuously supplying alkali-producing substrates and preventing strain death due to nutrient depletion; the long-lasting effects of humic acid and calcium carbonate can fix aluminum ions, preventing their reactivation. Unlike high-inoculation-rate liquid bacterial agents that suffer from pH decline and aluminum toxicity rebound in the later stages, this amendment maintains a stable soil pH above 5.5 for 60 days after application, with a continuous decrease in exchangeable aluminum content and no rebound. Simultaneously, the amendment process improves soil organic matter and aggregate structure, gradually enhancing soil buffering capacity, achieving a long-term stable amendment effect with a single application, significantly reducing the frequency and cost of amendments.

[0028] 5. The strains of this invention were isolated and screened from natural saline-alkali soils. As indigenous microorganisms, they pose no pathogenicity or ecological risk. All raw materials are environmentally friendly agricultural inputs, free of toxic or harmful components. Application will not cause soil compaction, salinization, or groundwater pollution, meeting the requirements of green agricultural development. The soil conditioner is suitable for various acidic soils such as red soil, lateritic red soil, and red soil, and can be widely applied in farmland, orchards, tea gardens, and woodlands. Application methods are flexible, including basal application, topdressing, and hole application, and it can also be used in combination with organic and chemical fertilizers. Furthermore, the microbial agent production process is simple, energy-efficient, and allows for large-scale production with controllable production costs. This technology achieves efficient soil improvement while balancing ecological safety and economic benefits, providing a green solution for sustainable agricultural development in acidic soil regions of southern my country. Attached Figure Description

[0029] Figure 1 This is a graph showing the pH measurement results of the fermentation broth of the strains obtained from soil screening in Dongying.

[0030] Figure 2 This is a graph showing the pH test results of the fermentation broth of the strain in an acidic environment of pH=5.5;

[0031] Figure 3 This is a graph showing the pH results of the fermentation broth of the strain over 7 consecutive days;

[0032] Figure 4 This is a graph showing the pH test results of soil samples containing strain D5-N19.

[0033] Figure 5 This is a colony diagram of strain D5-N19;

[0034] Figure 6 This is a Gram-stained image of strain D5-N19. Detailed Implementation

[0035] In this embodiment of the invention, an alkali-producing bacterium, *Providencia rettgeri*, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38612, on May 9, 2026. The colonies of this strain are nearly round, milky white, and have a moist, glossy surface. The strain is a Gram-positive bacillus.

[0036] The method for isolating and screening alkali-producing bacteria includes the following steps:

[0037] (1) Samples were collected from alkaline soil in the saline-alkali area of ​​Dongying, Shandong Province, and 10 samples were prepared. 4 -10 5 Series of diluted suspensions;

[0038] (2) Spread the diluted suspension on NA medium plates and incubate at 28°C for 48 hours. Select single colonies for purification three times.

[0039] (3) The purified single colony was inoculated into LB medium and cultured at 30°C for 48 hours. The pH value of the bacterial solution was measured to screen out potential alkali-producing bacteria that could increase the pH of the medium.

[0040] (4) Potential alkali-producing bacteria are inoculated into a compound liquid culture medium and cultured at 30°C and 200r / min for 48 hours. Strains that can significantly increase the pH of the culture medium in an acidic environment are screened out. The strains can increase the pH of the culture medium by more than 1.8 units when cultured in the compound liquid culture medium for 48 hours.

[0041] The composite liquid culture medium comprises the following raw materials (based on 1L volume) (see Table 1):

[0042] This culture medium is customized to the growth patterns and alkali-producing metabolic pathways of alkali-producing bacteria, achieving four functions: "gradient energy supply + continuous alkali production + acid tolerance acclimatization + enzyme activity enhancement".

[0043]

[0044] The preparation method of the compound liquid culture medium includes the following steps:

[0045] 1. Preparation of carbon source dispersion: Weigh soluble starch, add 5-10 times the mass of purified water at room temperature, stir to make a uniform, lump-free paste, then add glucose and sucrose in sequence, stir until initially dissolved, and obtain carbon source dispersion.

[0046] 2. Dissolving the basic nutrient solution: Weigh out tryptone, yeast extract powder, soybean meal powder, corn steep liquor powder, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, manganese sulfate, betaine, and sodium chloride. Add purified water, which accounts for 70%-80% of the total volume. Heat to 50-65℃ and stir for 20-40 minutes until the raw materials are completely dissolved to obtain the basic nutrient solution.

[0047] 3. Mixing and adjusting the volume: Slowly add the carbon source dispersion to the basic nutrient solution, stir continuously until well mixed, and add purified water to adjust the volume to the target volume.

[0048] 4. Initial pH adjustment: Use 0.5 mol / L dilute hydrochloric acid or sodium hydroxide solution to adjust the initial pH of the culture medium to 5.1-5.9.

[0049] 5. Dispensing and sterilization: Dispense the prepared culture medium into culture containers, seal them, and place them in a high-pressure steam sterilizer for sterilization at 114-121℃ for 15-25 minutes.

[0050] 6. Cooling and ready for use: After sterilization, slowly reduce the pressure and wait for the culture medium to cool naturally to 30-35℃ before taking it out and placing it in a sterile clean environment for later use.

[0051] The application of the alkali-producing bacteria in the improvement of acidic soil, wherein the acidic soil is red soil, lateritic red soil, or red soil, and the inoculation amount of the alkali-producing bacteria is 5%-10% of the soil weight.

[0052] A compound acid soil conditioner comprising alkali-producing bacteria and an agriculturally acceptable carrier.

[0053] The compound acid soil conditioner includes the following raw materials (by weight) (see Table 2):

[0054] This compound acidic soil conditioner uses alkali-producing bacteria as its core, constructing a four-dimensional system of "carrier protection - nutrient supply - buffer protection - synergistic improvement". The raw materials are as follows:

[0055]

[0056] A method for preparing a compound acidic soil conditioner includes the following steps:

[0057] 1. Carrier pretreatment: Peat, vermiculite powder, corn cob powder and wheat bran are crushed separately, passed through a 60-80 mesh standard sieve, mixed evenly according to the formula ratio, and placed in an autoclave at 121℃ for 20-30 minutes. After cooling to room temperature, they are ready for use.

[0058] 2. Preparation of nutrient matrix: Weigh corn flour, soybean meal, potassium humate, light calcium carbonate and fulvic acid according to the formula, mix them evenly, add an appropriate amount of purified water, stir until the moisture content is 30%-40% and the mixture is moistened, sterilize at 118℃ for 20 minutes, and cool for later use.

[0059] 3. Preparation of protective agent solution: Weigh trehalose, skim milk powder and sodium carboxymethyl cellulose according to the formula, add sterile purified water and stir until completely dissolved to prepare a protective agent aqueous solution with a mass concentration of 10%-15%. After filtration through a 0.22μm filter membrane for sterilization, it is ready for use.

[0060] 4. Adsorption and mixing of bacterial powder: Add the alkali-producing bacterial powder and the pretreated carrier into a horizontal mixer and stir at low speed for 10-20 minutes to initially mix them; then add the nutrient substrate to the mixer and continue stirring for 15 minutes; finally, spray the protective agent aqueous solution and continue stirring for 20-30 minutes until the material is uniform.

[0061] 5. Low-temperature drying: Spread the evenly mixed wet bacterial agent on a tray to a thickness of 1-2 cm, place it in a low-temperature ventilated drying oven, and dry it at 30-40℃ until the moisture content of the material is ≤20%.

[0062] 6. Finished product packaging: Crush the dried inoculant, pass it through a 40-mesh sieve, package it quantitatively according to specifications, seal it, and store it in a cool, dry place.

[0063] Technical principle of the invention:

[0064] 1. Principles of the preparation technology of compound liquid culture medium

[0065] (1) Functions and roles of each raw material

[0066] Glucose: a fast-acting small molecule carbon source that can be directly absorbed and utilized by the strain, providing energy rapidly for the lag phase and logarithmic growth phase after inoculation, and shortening the start-up time of the strain.

[0067] Sucrose: a medium-speed disaccharide carbon source that needs to be broken down by sucrase secreted by the strain before it can be utilized to maintain energy supply in the mid-to-late logarithmic growth phase and prolong the cell proliferation cycle.

[0068] Soluble starch: a long-lasting polysaccharide carbon source with a slow decomposition rate, which can continuously release glucose during the stable period of the strain, avoiding rapid depletion of nutrients that could lead to premature cell death and prolonging the duration of alkali production.

[0069] Tryptone: a high-purity animal-derived organic nitrogen source, rich in small molecule peptides and free amino acids, is the core substrate for alkalization by strains and directly determines the amount of alkalizing substances produced.

[0070] Yeast extract powder: Rich in B vitamins, nucleotides and trace elements, it acts as a growth factor to significantly enhance the metabolic activity of microorganisms, while also supplementing some organic nitrogen.

[0071] Soybean meal powder: a plant-derived long-lasting organic nitrogen source containing large molecular proteins and polypeptides, which need to be gradually decomposed and utilized by the proteases secreted by the strains, continuously supplying ammoniation substrates and extending the alkali production cycle.

[0072] Corn steep liquor powder: rich in amino acids, biotin, organic acids and minerals, which can strengthen the strain's stress-resistant metabolic pathways, improve the survival rate and metabolic activity under acid stress, and promote cell wall synthesis.

[0073] Dipotassium hydrogen phosphate: provides phosphorus and potassium, and forms a phosphate buffer system to alleviate drastic pH fluctuations during alkali production by the strain, maintaining a suitable environment for bacterial growth.

[0074] Magnesium sulfate: Magnesium ions are coenzyme components of many key enzymes in the strain, such as proteases, peptidases, and respiratory enzymes. They can significantly enhance the activity of alkali-producing enzyme systems and improve alkali production efficiency.

[0075] Calcium chloride: Calcium ions can stabilize the phospholipid bilayer structure of bacterial cell membranes, reduce the damage of hydrogen ions to cell membranes in acidic environments, and participate in the synthesis of peptidoglycan in cell walls, thereby enhancing the mechanical strength and acid resistance of bacteria.

[0076] Manganese sulfate: Manganese ions can activate the activity of carbonic anhydrase and dehydrogenase, enhance the respiratory alkalization and carbonate production pathways of the strain, and improve the alkalization efficiency per unit cell.

[0077] Betaine: A compatible osmotic protectant that can accumulate in bacterial cells, balance intracellular and extracellular osmotic pressure, protect the spatial structure of proteins and enzymes, and significantly enhance the acid stress tolerance of the strain.

[0078] Sodium chloride: Maintains the osmotic pressure balance of the culture medium, ensures the normal physiological function of the bacterial cell membrane, and avoids bacterial damage caused by osmotic pressure difference.

[0079] (2) Synergistic effect among raw materials

[0080] This culture medium achieves a simultaneous leap in strain growth rate, alkali production efficiency, and acid tolerance through the synergistic effect of multiple components:

[0081] Synergistic carbon source: Glucose (fast-acting) + sucrose (medium-acting) + soluble starch (long-acting) form a gradient release carbon source supply system, which perfectly matches the energy requirements of the strain in the lag phase, logarithmic phase and stationary phase. This avoids the excessive growth and premature death of the bacteria caused by a single fast-acting carbon source, and solves the problems of slow start-up and long lag phase caused by a single long-acting carbon source, thus extending the effective alkali production cycle of the strain from 72h to more than 120h.

[0082] Nitrogen source synergy: Tryptone (small molecule, fast-acting nitrogen) + soybean meal extract (large molecule, long-acting nitrogen) form a continuous and stable nitrogen supply, ensuring the nitrogen requirements of the strain during the proliferation phase and continuously providing substrate for ammoniation alkalization during the stationary phase, thus avoiding pH drop caused by nitrogen depletion during alkalization. Simultaneously, the carbon-to-nitrogen ratio is optimized to maintain it within the suitable range for strain growth and alkalization metabolism, preventing excessive carbon from leading to anabolism and insufficient alkalization, and also preventing excessive nitrogen from inhibiting strain growth.

[0083] Synergistic effect of minerals and stress resistance: Magnesium, manganese and calcium synergistically activate the key enzyme system for alkalization, while betaine provides osmotic protection, thereby enhancing the acid resistance of the strain at both the enzyme activity and cell structure levels. This can solve the technical problem of metabolic inhibition of strains under acid stress in conventional culture media.

[0084] (3) Necessity and importance of process parameter selection

[0085] Dissolving temperature 50-65℃: This temperature range accelerates the dissolution of macromolecular raw materials such as soybean meal powder and corn steep liquor powder, without destroying the protein nutrients and vitamin activity. Below 50℃, the raw materials dissolve slowly and incompletely, resulting in insufficient effective nutrients in the culture medium; above 65℃, protein denaturation and vitamin decomposition are likely to occur, leading to nutrient loss and decreased bacterial growth and alkali production.

[0086] Initial pH 5.1-5.9: Using a weakly acidic initial pH to acclimate the strain to acid tolerance can induce the expression of acid tolerance-related genes and alkali-producing enzyme systems, enabling the cultured strains to quickly adapt to the environment after inoculation into acidic soil. When the pH is below 5.1, the acid stress is too strong, the lag phase of the strain is greatly prolonged, and it may even fail to grow normally; when the pH is above 5.9, the acid tolerance acclimatization effect is insufficient, the strains have poor adaptability after entering the acidic environment, and the survival rate and alkali-producing capacity are significantly reduced.

[0087] Sterilization conditions: 114-121℃, 15-25min. These conditions thoroughly kill bacteria and spores in the culture medium while minimizing nutrient damage. Too low a temperature or too short a time will result in incomplete sterilization and easy contamination; too high a temperature or too long a time will trigger the Maillard reaction, causing sugars and proteins to caramelize and denature, reducing the nutrient value of the culture medium and hindering bacterial growth.

[0088] Cooling temperature 30-35℃: This is the optimal growth temperature for the matched strain. After inoculation, the strain can quickly initiate metabolism and enter the logarithmic growth phase. Inoculation at excessively high temperatures can lead to heat damage or even death of the bacteria; while inoculation at excessively low temperatures will decrease the metabolic activity of the strain and prolong the lag phase.

[0089] 2. Principles of the preparation technology of compound acidic soil conditioner

[0090] (1) Functions and roles of each raw material

[0091] Alkali-producing bacteria powder: The core functional component produces alkaline substances through ammonification, respiration, ion exchange, and carbonate formation, which neutralize soil acidity and reduce the activity of exchangeable aluminum.

[0092] Peat: A porous organic carrier rich in organic matter with strong adsorption capacity. It can fix bacterial cells, provide a micro-ecological shelter for bacterial strains, and at the same time increase the organic matter content of the soil and improve the soil structure.

[0093] Vermiculite powder: A layered porous mineral carrier with strong water and fertilizer retention capacity. It can improve soil permeability, slowly release mineral ions such as calcium and magnesium, and replenish soil basic ions.

[0094] Corn cob powder: a loose and porous plant-derived carrier that optimizes the aeration of microbial agents. After being applied to the soil, it slowly decomposes and releases carbon sources, providing continuous nutrition for the colonization of microbial strains.

[0095] Wheat bran: an organic carrier rich in crude fiber and B vitamins, serves as both an attachment substrate for microorganisms and a means to gradually decompose and release nutrients, supporting the long-term reproduction of bacterial strains.

[0096] Corn flour: a fast-acting carbon source that rapidly degrades into glucose after being applied to the soil, providing energy for the recovery and initial colonization of bacterial strains and helping them quickly form a dominant bacterial community.

[0097] Soybean meal powder: a long-lasting organic nitrogen source that gradually decomposes and releases small molecule peptides and amino acids, continuously providing substrates for the ammonia-based alkali production of the strain and extending the alkali production cycle.

[0098] Potassium humate: A high-molecular-weight organic amendment that can promote the formation of soil aggregates. At the same time, it can reduce aluminum toxicity by complexing soil aluminum ions through functional groups such as carboxyl and phenolic hydroxyl groups, thus synergistically improving the amendment effect.

[0099] Trehalose: a non-reducing disaccharide protectant that can replace water molecules and bind to bacterial proteins and phospholipids under dry and soil stress conditions, maintaining cell structure integrity and significantly improving strain resistance and survival rate.

[0100] Skim milk powder: a protein protectant that can form a protective film on the surface of bacteria, reducing acid stress and drying damage, and at the same time providing an initial nitrogen source for the strain after degradation.

[0101] Light calcium carbonate: a slightly soluble buffer that, when applied to the soil, can slowly neutralize the strong acid around the bacterial agent, creating a mild microenvironment for bacterial colonization and preventing the bacterial strain from dying rapidly due to strong acid stress when it is first introduced.

[0102] Sodium carboxymethyl cellulose: a water-soluble binder that improves the formability and stability of bacterial agent particles, reduces bacterial cell shedding and loss, and slowly degrades to provide a carbon source for the bacterial strain.

[0103] Fulvic acid: a small molecule active substance that can activate the metabolic enzyme activity of bacterial strains, improve alkali production efficiency, stimulate crop root growth, and chelate toxic metal ions in the soil.

[0104] (2) Synergistic effect among raw materials

[0105] This improver achieves a comprehensive enhancement of strain survival rate, action period, and improvement effect through multi-component synergy:

[0106] Carrier synergy: Peat (organic adsorption) + vermiculite (mineral water retention) + corn cob (loose and breathable) + bran (slow release of nutrients) form a multi-level porous carrier network, which not only provides the bacteria with a microenvironment to avoid strong acids, aluminum poisoning and protozoan predation, but also retains water and fertilizer and continuously releases nutrients, extending the survival period of the strain in the soil from 15 days to more than 60 days.

[0107] Synergistic nutrition: Corn flour (quick-acting carbon) and soybean meal (long-acting nitrogen) form a gradient nutrient supply. In the early stage of application, quick-acting carbon provides energy quickly and helps the strain recover and colonize rapidly. In the middle and late stages, long-acting nitrogen is continuously released, providing a stable substrate for ammoniation and alkali production, which solves the problem of nutrient deficiency and rapid death after the application of a single bacterial agent.

[0108] Synergistic Protection: Light calcium carbonate (microenvironment buffer) + trehalose (cell structure protection) + skim milk powder (outer layer protection) form a triple protection mechanism: calcium carbonate first neutralizes local acidity, reducing initial stress; trehalose and skim milk powder protect the bacterial cell structure and enhance stress resistance. The synergistic effect of these three factors significantly improves the survival rate of the strain in suitable acidic soil.

[0109] Synergistic Functions: The bacterial strain produces alkali, calcium carbonate acts as a chemical buffer, and humic acid / fulvic acid complexes to fix aluminum. These three actions work simultaneously to neutralize acidity, alleviate aluminum toxicity, and improve soil fertility. The bacterial strain is responsible for long-term biological improvement, calcium carbonate provides rapid buffering, and humic acid enhances soil fertility. These three elements complement each other, alleviating the short-lived effects of chemical improvement and compensating for the slow results of single-microbial improvement.

[0110] (3) Necessity and importance of process parameter selection

[0111] Carrier particle size 60-80 mesh: Carriers in this particle size range have a moderate specific surface area, strong adsorption capacity for bacteria, and good dispersibility and aeration after being applied to the soil. If the particle size is too large, the specific surface area is small, the amount of bacteria adsorbed is small, and the uniformity of the inoculant is poor; if the particle size is too small, it is easy to clump, has poor aeration, and is easily washed away by water after being applied to the soil.

[0112] Sterilization conditions: 121℃, 20-30 min: Thoroughly kill miscellaneous bacteria, insect eggs, and weed seeds in the carrier and nutrient substrate, preventing miscellaneous bacteria from competing with functional bacteria for nutrients. Insufficient sterilization time can lead to incomplete sterilization and contamination of the inoculant during storage; excessive time can cause nutrient charring, reducing the carrier's nutrient supply capacity.

[0113] Drying temperature 30-40℃: Low-temperature drying can retain the activity of bacteria to the greatest extent while controlling the moisture content. If the temperature is below 30℃, the drying efficiency is low, the production cycle is long, and the moisture content is prone to exceed the standard, leading to mold growth; if the temperature is above 40℃, the bacterial protein is prone to denaturation, the number of viable bacteria drops significantly, and the bacterial agent becomes ineffective.

[0114] Moisture content ≤20%: At this moisture content, the bacterial agent has good storage stability, is not prone to the growth of miscellaneous bacteria, and the bacteria remain in a low metabolic dormant state. If the moisture content is too high, the bacterial agent is prone to clumping and mold growth, and the number of viable bacteria will rapidly decline; if the moisture content is too low, the bacteria will enter deep dormancy, resulting in slow recovery after application to the soil and delayed effectiveness.

[0115] This technology revolves around a complete technology chain: screening acid-resistant and alkali-producing strains, optimizing cultivation to enhance performance, preparing inoculants to ensure colonization, and synergistic and long-term soil improvement. It achieves systemic innovation from four levels, and its core principles are as follows:

[0116] 3. Strain screening and functional principles

[0117] A strain capable of stably producing alkali under acidic conditions was obtained from alkaline soil in Dongying, Shandong Province, through a step-by-step screening strategy involving gradient dilution, plate purification, and initial and secondary screening. This strain primarily produces alkali through four pathways: first, secreting proteases and peptidases to decompose organic nitrogen and produce ammonia, which dissolves in water to form ammonium hydroxide to neutralize hydrogen ions; second, aerobic respiration consumes carbon dioxide, reducing carbonic acid concentration and decreasing hydrogen ion dissociation; third, hydrogen ions are transported intracellularly via H+-ATPase in the cell membrane, releasing basic cations; and fourth, carbonic anhydrase catalyzes the hydration of carbon dioxide to generate bicarbonate, which is released extracellularly via a transport system and combines with soil cations to form carbonate-based alkaline buffers. Simultaneously, extracellular polysaccharides and proteins produced by the strain's metabolism can complex aluminum ions, and cell wall peptidoglycans can adsorb aluminum ions, thus mitigating aluminum toxicity.

[0118] 4. Principles of Culture Medium Enhancement and Regulation

[0119] The specialized compound liquid culture medium enhances the alkali-producing performance and acid tolerance of the strains through a dual mechanism of nutrient regulation and stress acclimatization. By precisely proportioning a tertiary carbon source and a compound nitrogen source, the carbon-nitrogen ratio is optimized to the best balance point between strain growth and alkali production, ensuring rapid cell proliferation while inducing high expression of alkali-producing enzymes. The addition of mineral elements such as magnesium, manganese, and calcium activates the activity of key alkali-producing enzymes, increasing the alkali production efficiency per cell. The addition of betaine and a weakly acidic initial pH acclimatization induces the expression of acid-tolerant genes in the strains, enhancing their intracellular osmotic pressure protection and enabling them to maintain high metabolic activity in acidic environments.

[0120] 5. Principle of Microbial Colonization and Protection

[0121] This compound acidic soil conditioner creates a suitable microenvironment to ensure the survival and colonization of bacterial strains in acidic soils. The porous composite carrier provides physical protection, isolating the strains from the strong acid environment and protozoan predation; buffer components rapidly regulate the micro-pH around the inoculant, reducing initial acid stress; protectants maintain the stability of bacterial cell structure, enhancing resilience; and gradient nutrient components continuously release nutrients, supporting bacterial reproduction and metabolism. These multiple synergistic effects enable the strains to successfully overcome the initial environmental stress after application, rapidly colonize to form a dominant bacterial community, and continuously exert their soil-improving effects. At high inoculum levels, the carrier can immobilize excess bacterial cells, preventing concentrated cell death and decomposition that releases organic acids, thus fundamentally mitigating the pH drop problem associated with high-inoculum liquid inoculants.

[0122] 6. Principles of Comprehensive Soil Improvement

[0123] When compound acid soil conditioner is applied to acidic soil, it achieves a tiered comprehensive improvement through multiple synergistic mechanisms: In the rapid phase (0-7 days), calcium carbonate and humic acid quickly buffer acidity and complex aluminum ions, alleviating soil acidification damage in the short term and creating a mild microenvironment for bacterial colonization; in the intermediate phase (7-30 days), the bacterial strains rapidly recover and colonize, continuously producing alkali to increase soil pH and reduce exchangeable aluminum content, becoming the main force in soil improvement; in the long-term phase (30-60 days and beyond), the continuous metabolism of the bacterial strains and the decomposition of the carrier release nutrients, promoting the growth of beneficial soil microorganisms, increasing soil organic matter content and aggregate structure, and enhancing the soil's own buffering capacity. Ultimately, this achieves a complete improvement pathway of "rapid mitigation - intermediate improvement - long-term fertilization," fundamentally improving the physicochemical and biological properties of acidic soil.

[0124] To make the present invention more fully disclosed, more specific embodiments are described below.

[0125] Example 1

[0126] 1. Experimental Materials and Procedures

[0127] 1.1 Microbial Culture Media

[0128] NA medium: 10g peptone, 3g beef extract, 5g sodium chloride, 18g agar, add distilled water to a final volume of 1L.

[0129] PDA medium: 200g peeled potato, 20g glucose, 18g agar, pH 6.5, add distilled water to a final volume of 1L.

[0130] LB medium: 10 g peptone, 10 g sodium chloride, 5 g yeast extract, pH 7.0, add distilled water to a final volume of 1 L.

[0131] Compound liquid culture medium (1L): glucose 9g, sucrose 4g, soluble starch 3g, tryptone 12g, yeast extract powder 5g, soybean meal powder 4g, corn steep liquor powder 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.5g, calcium chloride 0.1g, manganese sulfate 0.03g, betaine 1.0g, sodium chloride 5g.

[0132] The preparation method of the compound liquid culture medium includes the following steps:

[0133] 1. Preparation of carbon source dispersion: Weigh soluble starch, add 8 times the mass of purified water at room temperature, stir to make a uniform paste without lumps, then add glucose and sucrose in sequence, stir until initially dissolved, and obtain carbon source dispersion.

[0134] 2. Dissolving the basic nutrient solution: Weigh out tryptone, yeast extract powder, soybean meal powder, corn steep liquor powder, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, manganese sulfate, betaine, and sodium chloride. Add purified water, which accounts for 750% of the total volume. Heat to 60°C and stir for 30 minutes until the raw materials are completely dissolved to obtain the basic nutrient solution.

[0135] 3. Mixing and adjusting the volume: Slowly add the carbon source dispersion to the basic nutrient solution, stir continuously until well mixed, and add purified water to adjust the volume to the target volume.

[0136] 4. Initial pH adjustment: Use 0.5 mol / L dilute hydrochloric acid solution to adjust the initial pH of the culture medium to 5.5.

[0137] 5. Dispensing and sterilization: Dispense the prepared culture medium into culture containers, seal them, and place them in a high-pressure steam sterilizer for sterilization at 118°C for 20 minutes.

[0138] 6. Cooling and storing: After sterilization, slowly reduce the pressure and allow the culture medium to cool naturally to 32°C. Then, remove it and store it in a sterile clean environment for later use.

[0139] PDB medium: 200 g peeled potato, 20 g glucose, pH 6.5, add distilled water to a final volume of 1 L.

[0140] Potato juice preparation: Peel 200 g of fresh potatoes and cut them into small pieces. Add 1000 mL of distilled water and boil for 20-30 minutes, until the potatoes are soft. Filter through multiple layers of gauze and collect the filtrate.

[0141] 1.2 Isolation and Identification of Alkali-Producing Microorganisms

[0142] Soil samples were collected from Dongying, Shandong Province, for the isolation and identification of alkali-producing bacteria. First, 10 g of soil was weighed and dispersed in sterile water by shaking for 2-3 hours, and a series of Xishi suspensions (10 g / kg) were prepared. 4 -10 5 100 μL of each culture was spread onto NA agar plates to screen for alkali-producing bacteria. The NA plates were then incubated at 28°C for 48 h. Approximately 100 μL of each culture was spread onto PDA agar plates to screen for alkali-producing fungi. The PDA plates were then incubated at 30°C for 96 h. Single colonies of different morphologies were selected from the plates at the maximum dilution and isolated and purified on both NA and PDA plates. Pure single colonies were obtained after three purification processes. Bacterial single colonies were then inoculated into LB agar and incubated at 30°C for 48 h; fungal single colonies were inoculated into PDB liquid agar and incubated at 30°C for 96 h. The pH of the bacterial cultures was measured, and strains that could raise the pH of the culture medium were identified as potential alkali-producing bacteria. These potential alkali-producing bacteria were inoculated into a compound liquid agar medium at pH 5.5 and incubated in a constant-temperature shaker at 30°C and 200 rpm for 48 h to screen for strains that could raise the pH of the culture medium in an acidic environment.

[0143] 1.3 Identification of the alkali-producing ability of the strain

[0144] Select the purified single colonies and inoculate them into a compound liquid culture medium for resuscitation and culture. Incubate at 30°C and 200 rpm for 24 hours to allow the bacteria to enter the mid-log phase. The bacterial concentration OD 600 =1.0±0.05, which is the seed culture. The seed culture was then inoculated into the compound liquid culture medium at a volume ratio of 1%. The culture was incubated on a shaker at 30℃ for 7 days, and the pH and OD values ​​of the bacterial culture were measured at 0, 24, 48, 72, 120, and 168 h. Uninoculated compound liquid culture medium and PDB liquid culture medium served as blank controls.

[0145] 1.4 Soil pot culture experiment

[0146] The soil samples were collected from a sugarcane field in Cendui Village, Setuan Town, Jiangzhou District, Chongzuo City, Guangxi Zhuang Autonomous Region. The soil was air-dried and passed through a 2 mm sieve. The soil pH was 4.54, organic matter was 2.46%, total phosphorus was 0.19%, total potassium was 0.33%, and total nitrogen was 1.45%.

[0147] The selected alkali-producing strains were inoculated into a compound liquid culture medium at a 1% inoculum and cultured in a constant-temperature shaker at 30℃ and 200 r / min for 24 h to prepare a seed culture. Then, the seed culture was inoculated into a fresh compound liquid culture medium at a 2% inoculum and cultured in a constant-temperature shaker at 30℃ and 200 r / min for 24 h. The OD was then measured. 600 OD 600 =1.0±0.05 was used as the inoculum.

[0148] Four treatments were performed: ① An equal volume of sterile water was added as a blank control; ② 1.5 kg of soil per pot was inoculated with a cultured Alkali-producing bacteria solution at a rate of 5% of the soil weight, with two replicates for each treatment; ③ 1.5 kg of soil per pot was inoculated with a cultured Alkali-producing bacteria solution at a rate of 8% of the soil weight, with two replicates for each treatment; ④ 1.5 kg of soil per pot was inoculated with a cultured Alkali-producing bacteria solution at a rate of 10% of the soil weight, with two replicates for each treatment. The plants were naturally incubated, observed and watered daily to maintain soil moisture, and soil pH was measured periodically.

[0149] 2. Experimental Results

[0150] 2.1 Purification and screening results

[0151] use pH Calculation of shake flask culture 48h Preliminary measurements were performed on the culture medium, and the results are shown in the table. 3 and Figure 1 A total of 69 bacterial strains were screened, and 24 strains were found to increase the pH of LB medium by more than 0.5 units compared with the blank control.

[0152]

[0153]

[0154] (Note: CK represents the blank control group)

[0155] 2.2 Screening of alkali-producing bacteria in acidic environments

[0156] See Table 4 and Figure 2 In the compound liquid culture medium with pH=5.5, five strains showed a significant increase in pH compared to the blank control group. Among them, the strain with the largest increase in pH was D4-N6 (pH 7.48), which increased by 1.89 units after 48 hours of culture compared to the blank control group (pH 5.58).

[0157]

[0158] (Note: CK represents the blank control group)

[0159] 2.3 Results of the identification of the alkali-producing ability of the strain

[0160] The results of 7 consecutive days of culture and testing (see Table 5 and...) Figure 3 The study found that strain D5-N19 had the best alkali-producing ability and was identified as the target functional strain for this pot experiment.

[0161]

[0162] (Note: CK represents the blank control group)

[0163] 2.4 Results of Soil Pot Culture Experiment

[0164] (1) The selected strain D5-N19 was inoculated into a compound liquid culture medium at an inoculation rate of 1%, and cultured at 30℃ and 200 r / min for 24 h to prepare a seed culture. Then, the seed culture was inoculated into a compound liquid culture medium at an inoculation rate of 5% by volume for expansion culture, and cultured at 30℃ and 200 r / min for 12-24 h. The OD was measured. 600 OD 600 When the concentration is 1.0, it is used as the inoculum.

[0165] (2) Inoculate the prepared bacterial culture at 5%, 8%, and 10% of the soil weight, with two replicates for each treatment. Incubate at room temperature for 60 days, maintaining soil moisture by adding water. Regularly collect soil samples to measure pH according to NY / T 1121.2-2006 standard. Results are shown in Table 6. Figure 4 .

[0166]

[0167] (Note: CK represents the blank control group)

[0168] As shown in Table 6:

[0169] (1) Significant short-term (0-20 d) pH increase effect

[0170] Soil pH in all treatment groups showed a rapid upward trend in the early stage of cultivation (3-20 days). On day 3 of cultivation, the initial pH values ​​of each group ranged from 4.41 to 4.48, with no significant difference. By day 20, the pH of the CK group rose to 5.29, while the pH values ​​of the 5%, 8%, and 10% strain D5-N19 treatment groups reached 5.39, 5.38, and 5.45, respectively, all significantly higher than the control group. Moreover, the peak pH value increased with the increase of inoculum size, indicating that strain D5-N19 can effectively improve the pH of acidic soil in the short term, and the short-term improvement effect is better with a higher inoculum size.

[0171] (2) Significant pH fluctuations during the middle period (21-30 days)

[0172] During the 20-30 day period, the pH of the CK group showed a slow upward trend with small fluctuations; while the pH of the D5-N19 treatment group showed significant fluctuations: the 8% treatment group had a second pH peak (5.54) on day 23, which then fell back; the 10% treatment group showed a significant decrease on day 23 (pH dropped to 4.68), indicating that the metabolic activity of the strain may have produced complex acid-base reactions in the middle period, leading to a decrease in pH stability.

[0173] (3) Long-term (30-60 days) improvement effect reversed, high inoculum exacerbated pH drop.

[0174] During the later stages of cultivation (30-60 days), the pH of the CK group rose slowly and steadily, reaching 5.34 on day 60, the highest among all treatment groups. In contrast, the pH of the D5-N19 treatment groups showed a continuous downward trend, with the decrease being greater at higher inoculum levels. By day 60, the pH values ​​of the 5%, 8%, and 10% treatment groups were 5.01, 4.765, and 4.735, respectively, all significantly lower than the CK group. The pH values ​​of the 8% and 10% treatment groups had already returned to near their initial levels, indicating that the short-term pH-raising effect of strain D5-N19 is not sustainable in the long term, and high inoculum levels may even exacerbate soil acidification in the later stages.

[0175] In summary, the pH-improving effect of strain D5-N19 on acidic soil exhibits a clear time- and dose-dependent effect: it can effectively raise soil pH in the short term (≤20 days), but the effect is unstable in the long term (>30 days), and high inoculum amounts can actually lead to a continuous decline in soil pH. The 5% inoculum treatment group still had a pH (5.01) higher than the initial level at 60 days, and the decline was the smallest, demonstrating relatively better long-term stability in bacterial suspension treatment, which can serve as a reference for subsequent optimization of inoculum amounts.

[0176] 2.5 Results of Soil Pot Tests on Exchangeable Acid, Exchangeable Hydrogen, and Exchangeable Aluminum

[0177] (1) The selected strain D5-N19 was inoculated into a compound liquid culture medium at an inoculation rate of 1%, and cultured at 30℃ and 200 r / min for 24 h to prepare a seed culture. Then, the seed culture was inoculated into a compound liquid culture medium at an inoculation rate of 5% by volume for expansion culture, and cultured at 30℃ and 200 r / min for 12-24 h. The OD was measured. 600 OD 600 When the concentration is 1.0, it is used as the inoculum.

[0178] (2) The cultured bacterial solution was added at an inoculum amount of 5%, 8%, and 10% of the soil weight, and each treatment was replicated twice. The culture was carried out at room temperature for 60 days, and the soil moisture was maintained by adding water during the period. The soil was sampled every 30 days to test the exchangeable hydrogen and exchangeable aluminum index. The results are shown in Table 7.

[0179] The detection methods for exchangeable hydrogen and exchangeable aluminum are as follows: The contents of exchangeable acid, exchangeable hydrogen and exchangeable aluminum are determined according to the method of potassium chloride extraction-titration for the determination of exchangeable acidity of soil (HJ 649-2013).

[0180] Exchangeable acidity: The total amount of hydrogen and aluminum ions in the soil that can be exchanged with neutral salts. It is a direct indicator of soil acidity. The lower the value, the weaker the soil acidity.

[0181] Exchangeable hydrogen / aluminum: In acidic soils, exchangeable aluminum is the main source of acidification (aluminum ion hydrolysis produces a large number of hydrogen ions), and reducing exchangeable aluminum is a key goal for improving acidic soils.

[0182] Exchangeable aluminum difference (30d-60d): The larger the difference, the more significant the decrease in soil exchangeable aluminum within 30-60 days of cultivation, and the stronger the mitigation effect of the strain on aluminum toxicity; if the difference is negative, it means that the exchangeable aluminum has increased instead of decreased, and the strain has lost its improvement effect.

[0183]

[0184] (Note: CK represents the blank control group)

[0185] As shown in Table 7:

[0186] (1) Short-term effect after 30 days: The exchangeable aluminum in all strain D5-N19 treatment groups was lower than that in the CK group (9.02-9.89 vs 12.3), indicating that the strain can effectively reduce soil exchangeable aluminum and alleviate aluminum toxicity in the short term.

[0187] (2) Differentiation of long-term effects over 60 days:

[0188] The 5% strain D5-N19 group: exchangeable aluminum decreased to 2.85 mmol / kg, which was lower than 3.64 in the CK group. It was the only bacterial suspension treatment group that had a better long-term effect than the control group, indicating that the low inoculum of strain D5-N19 can continuously reduce exchangeable aluminum and has a stable improvement effect.

[0189] In the 8% and 10% strain D5-N19 groups, the exchangeable aluminum increased to 7.53 mmol / kg and 10.07 mmol / kg respectively after 60 days, which was much higher than that of the CK group. The 10% group even exceeded the level after 30 days, indicating that high inoculation of strain D5-N19 can lead to a rebound in soil exchangeable aluminum in the later stage, exacerbating the risk of aluminum toxicity.

[0190] 2.6 Identification of strain D5-N19

[0191] ① Morphological identification, such as Figure 5 As shown: Strain D5-N19 was inoculated on LB agar plates and cultured for 24 h. The colonies were nearly round, milky white, and moist and glossy. ② After extracting and sequencing the strain's DNA, molecular identification was performed. The DNA sequence of the strain was amplified, and the 16S region was amplified and sequenced by PCR. The primers used were: forward sequence 27F: 5-AGAGTTTGATCCTGGCTCAG-3, reverse sequence 1492R: 5TACGGCTACCTTGTTACGACTT-3'. After sequencing the PCR product, the obtained 16S sequence is shown in SEQ ID NO: 1 of the sequence listing. BLAST alignment and housekeeping gene detection confirmed that this strain is closely related to *Providencia rettgeri*. Based on morphological identification, the strain was classified and named *Providencia rettgeri*. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38612, and the deposit date is May 9, 2026.

[0192] 2.7 Gram staining results of strain D5-N19

[0193] like Figure 6 As shown, strain D5-N19, after Gram staining, has a purple-red rod-shaped cell, indicating it is a Gram-positive bacillus.

[0194] 2.8 Application of strain D5-N19

[0195] The compound acidic soil conditioner, by weight, includes the following raw materials: 10 parts D5-N19 bacterial powder, 28 parts peat, 15 parts vermiculite powder, 15 parts corn cob powder, 12 parts wheat bran, 6 parts corn flour, 5 parts soybean meal powder, 4 parts potassium humate, 2 parts trehalose, 2 parts skim milk powder, 5 parts light calcium carbonate, 0.5 parts sodium carboxymethyl cellulose, and 1.5 parts fulvic acid.

[0196] A method for preparing a compound acidic soil conditioner includes the following steps:

[0197] 1. Carrier pretreatment: Peat, vermiculite powder, corn cob powder and wheat bran are crushed separately, passed through a 70-mesh standard sieve, mixed evenly according to the formula ratio, and placed in an autoclave at 121℃ for 25 minutes. After cooling to room temperature, they are ready for use.

[0198] 2. Preparation of nutrient matrix: Weigh corn flour, soybean meal, potassium humate, light calcium carbonate and fulvic acid according to the formula, mix them evenly, add an appropriate amount of purified water, stir until the moisture content is 33% and the mixture is moistened, sterilize at 118℃ for 20 minutes, and cool for later use.

[0199] 3. Preparation of protective agent solution: Weigh trehalose, skim milk powder and sodium carboxymethyl cellulose according to the formula, add sterile purified water and stir until completely dissolved to prepare a protective agent aqueous solution with a mass concentration of 12%. After filtration through a 0.22μm filter membrane for sterilization, it is ready for use.

[0200] 4. Adsorption and mixing of bacterial powder: D5-N19 bacterial powder and pretreated carrier are put into a horizontal mixer and stirred at low speed for 15 minutes to initially mix. Then, the nutrient matrix is ​​added to the mixer and stirred for another 15 minutes. Finally, the protective agent aqueous solution is sprayed and stirred for another 25 minutes until the material is uniform.

[0201] 5. Low-temperature drying: Spread the evenly mixed wet bacterial agent on a tray to a thickness of 1.6 cm, place it in a low-temperature ventilated drying oven, and dry it at 35°C until the moisture content of the material is 18%.

[0202] 6. Finished product packaging: Crush the dried inoculant, pass it through a 40-mesh sieve, package it quantitatively according to specifications, seal it, and store it in a cool, dry place.

[0203] Application: Apply compound acidic soil conditioner at 5% of the soil weight.

[0204] 2.9 Single-factor screening experiment for key process parameters of compound liquid culture medium

[0205] The bacterial concentration (OD) of strain D5-N19 after 48 hours of culture 600 The evaluation index included the pH increase of the culture medium. Six key process parameters were selected for single-factor experiments. Each experiment was repeated three times, and the average value was taken.

[0206] Experiment 1: Initial pH Screening

[0207] The initial pH gradient was set to 4.7, 5.1, 5.5, 5.9, 6.3, and 6.7. The remaining parameters were consistent with the process in Example 1. The test results are shown in Table 8.

[0208]

[0209] Conclusion Analysis:

[0210] Below the optimal range (pH < 5.1): The environmental hydrogen ion concentration is too high, the permeability of the bacterial cell membrane increases, the activity of intracellular enzymes is inhibited, and the growth of the strain and alkali production metabolism are significantly inhibited. The OD value and the alkali production amplitude decrease rapidly as the pH decreases.

[0211] Greater than the optimal range (pH>5.9): The acid tolerance acclimatization effect disappears, the expression level of alkali-producing enzyme system of the strain is downregulated, and the initial pH is too high, which compresses the space for alkali production improvement, and the pH increase rate decreases significantly with the increase of initial pH.

[0212] Optimal range: 5.1-5.9; Best: 5.5.

[0213] Experiment 2: Screening of raw material dissolution temperature

[0214] The dissolution temperature gradient was set to 45, 50, 55, 60, 65, and 70°C. The remaining parameters were the same as those in Example 1. The test results are shown in Table 9.

[0215]

[0216] Conclusion Analysis:

[0217] Below the optimal range (<50℃): macromolecular raw materials are not fully dissolved, there are insufficient available nutrients in the culture medium, and the strain lacks sufficient substrate for growth and alkali production, resulting in poor performance.

[0218] Temperatures above the optimal range (>65℃): High temperatures cause the denaturation and inactivation of heat-sensitive nutrients, reduce the content of growth factors, decrease the metabolic activity of the strain, and reduce the growth and alkali production effects.

[0219] Preferred range: 50-65℃; Optimal range: 60℃.

[0220] Experiment 3: Sterilization Temperature Screening

[0221] The sterilization temperature gradient was set to 110, 114, 118, 121, 125, and 130°C. The remaining parameters were the same as those in Example 1. The test results are shown in Table 10.

[0222]

[0223] Conclusion Analysis:

[0224] Temperatures below the optimal range (<114℃): Incomplete sterilization results in residual bacterial spores competing for nutrients with functional bacteria, inhibiting the growth of functional bacteria and reducing the alkali production effect.

[0225] Above the optimal range (>121℃): High temperatures exacerbate the Maillard reaction, causing caramelization of sugars and proteins, extensive destruction of nutrients, and the generation of antibacterial byproducts, resulting in a significant decrease in the growth and alkali-producing capacity of bacterial strains.

[0226] Preferred range: 114-121℃; Optimal range: 118℃.

[0227] Experiment 4: Sterilization Time Screening

[0228] The sterilization time gradients were set to 10, 15, 20, 25, 30, and 35 min, with the remaining parameters consistent with the process in Example 1. The test results are shown in Table 11.

[0229]

[0230] Conclusion Analysis:

[0231] Less than the optimal range (<15min): Insufficient sterilization time, heat-resistant spores cannot be completely killed, and contamination by other bacteria is likely to occur in the later stages of culture, interfering with the normal growth and metabolism of functional bacteria.

[0232] Greater than the optimal range (>25 min): Prolonged high temperature leads to continuous degradation of nutrients, reduced nutrient potency of the culture medium, insufficient substrate for bacterial growth and alkali production, and a gradual decline in effectiveness.

[0233] Optimal range: 15-25 min; best range: 20 min.

[0234] Experiment 5: Screening of total carbon source concentration

[0235] The total carbon source concentration gradient was set to 8, 12, 16, 20, 24, and 28 g / L. The remaining parameters were the same as those in Example 1. The test results are shown in Table 12.

[0236]

[0237] Conclusion Analysis:

[0238] Less than the optimal range (<12g / L): Insufficient carbon source supply, strain energy deficiency, limited cell proliferation, low cell count, and insufficient total alkali production.

[0239] Excessive carbon source leads to an imbalance in the carbon-nitrogen ratio, causing the strain to proliferate excessively due to anabolism, inhibiting the ammonification-based alkalization metabolic pathway, and reducing the alkalization efficiency per unit cell mass.

[0240] Optimal range: 12-20 g / L; best range: 16 g / L.

[0241] Experiment 6: Screening of Betaine Dosage

[0242] The betaine addition gradient was set at 0, 0.5, 1.0, 1.5, 2.0, and 2.5 g / L. The remaining parameters were the same as those in Example 1. The test results are shown in Table 13.

[0243]

[0244] Conclusion Analysis:

[0245] Less than the optimal range (<0.5 g / L): Insufficient accumulation of intracellular compatibility solutes, easy denaturation of cell protein and enzyme structures under acid stress, low survival rate of strains, and inhibition of growth and alkali production.

[0246] Excessive betaine (>2.0 g / L) leads to increased osmotic pressure in the culture medium, water loss in bacterial cells, decreased metabolic activity, and the strain needs to consume additional energy to regulate osmotic pressure, resulting in reduced energy for growth and alkali production.

[0247] Preferred range: 0.5-2.0 g / L; Optimal range: 1.0 g / L.

[0248] Example 2

[0249] Compound liquid culture medium (1L): glucose 5g, sucrose 3g, soluble starch 2g, tryptone 8g, yeast extract powder 3g, soybean meal powder 2g, corn steep liquor powder 1g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.2g, calcium chloride 0.05g, manganese sulfate 0.01g, betaine 0.5g, sodium chloride 3g.

[0250] The preparation method of the compound liquid culture medium includes the following steps:

[0251] 1. Preparation of carbon source dispersion: Weigh soluble starch, add 8 times the mass of purified water at room temperature, stir to make a uniform paste without lumps, then add glucose and sucrose in sequence, stir until initially dissolved, and obtain carbon source dispersion.

[0252] 2. Dissolving the basic nutrient solution: Weigh out tryptone, yeast extract powder, soybean meal powder, corn steep liquor powder, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, manganese sulfate, betaine, and sodium chloride. Add purified water, which accounts for 750% of the total volume. Heat to 55°C and stir for 30 minutes until the raw materials are completely dissolved to obtain the basic nutrient solution.

[0253] 3. Mixing and adjusting the volume: Slowly add the carbon source dispersion to the basic nutrient solution, stir continuously until well mixed, and add purified water to adjust the volume to the target volume.

[0254] 4. Initial pH adjustment: Adjust the initial pH of the culture medium to 5.1 using 0.5 mol / L dilute hydrochloric acid or sodium hydroxide solution.

[0255] 5. Dispensing and sterilization: Dispense the prepared culture medium into culture containers, seal them, and place them in a high-pressure steam sterilizer for sterilization at 116°C for 18 minutes.

[0256] 6. Cooling and ready for use: After sterilization, slowly reduce the pressure and wait for the culture medium to cool naturally to 32°C before taking it out and placing it in a sterile clean environment for later use.

[0257] The compound acidic soil conditioner, by weight, includes the following raw materials: 5 parts D5-N19 bacterial powder, 20 parts peat, 10 parts vermiculite powder, 10 parts corn cob powder, 8 parts wheat bran, 3 parts corn flour, 2 parts soybean meal powder, 2 parts potassium humate, 1 part trehalose, 1 part skim milk powder, 3 parts light calcium carbonate, 0.1 parts sodium carboxymethyl cellulose, and 0.5 parts fulvic acid.

[0258] A method for preparing a compound acidic soil conditioner includes the following steps:

[0259] 1. Carrier pretreatment: Peat, vermiculite powder, corn cob powder and wheat bran are crushed separately, passed through a 60-mesh standard sieve, mixed evenly according to the formula ratio, and placed in an autoclave at 121℃ for 25 minutes. After cooling to room temperature, they are ready for use.

[0260] 2. Preparation of nutrient matrix: Weigh corn flour, soybean meal, potassium humate, light calcium carbonate and fulvic acid according to the formula, mix them evenly, add an appropriate amount of purified water, stir until the moisture content is 33% and the mixture is moistened, sterilize at 118℃ for 20 minutes, and cool for later use.

[0261] 3. Preparation of protective agent solution: Weigh trehalose, skim milk powder and sodium carboxymethyl cellulose according to the formula, add sterile purified water and stir until completely dissolved to prepare a protective agent aqueous solution with a mass concentration of 12%. After filtration through a 0.22μm filter membrane for sterilization, it is ready for use.

[0262] 4. Adsorption and mixing of bacterial powder: D5-N19 bacterial powder and pretreated carrier are put into a horizontal mixer and stirred at low speed for 15 minutes to initially mix. Then, the nutrient matrix is ​​added to the mixer and stirred for another 15 minutes. Finally, the protective agent aqueous solution is sprayed and stirred for another 25 minutes until the material is uniform.

[0263] 5. Low-temperature drying: Spread the evenly mixed wet bacterial agent on a tray to a thickness of 1.6 cm, place it in a low-temperature ventilated drying oven, and dry it at 30°C until the moisture content of the material is 20%.

[0264] 6. Finished product packaging: Crush the dried inoculant, pass it through a 40-mesh sieve, package it quantitatively according to specifications, seal it, and store it in a cool, dry place.

[0265] Application: Apply compound acidic soil conditioner at 5% of the soil weight.

[0266] Example 3

[0267] Compound liquid culture medium (1L): glucose 15g, sucrose 8g, soluble starch 6g, tryptone 18g, yeast extract powder 8g, soybean meal powder 7g, corn steep liquor powder 5g, dipotassium hydrogen phosphate 4g, magnesium sulfate 1.0g, calcium chloride 0.3g, manganese sulfate 0.08g, betaine 2.0g, sodium chloride 8g.

[0268] The preparation method of the compound liquid culture medium includes the following steps:

[0269] 1. Preparation of carbon source dispersion: Weigh soluble starch, add 8 times the mass of purified water at room temperature, stir to make a uniform paste without lumps, then add glucose and sucrose in sequence, stir until initially dissolved, and obtain carbon source dispersion.

[0270] 2. Dissolving the basic nutrient solution: Weigh out tryptone, yeast extract powder, soybean meal powder, corn steep liquor powder, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, manganese sulfate, betaine, and sodium chloride. Add purified water, which accounts for 750% of the total volume. Heat to 65°C and stir for 30 minutes until the raw materials are completely dissolved to obtain the basic nutrient solution.

[0271] 3. Mixing and adjusting the volume: Slowly add the carbon source dispersion to the basic nutrient solution, stir continuously until well mixed, and add purified water to adjust the volume to the target volume.

[0272] 4. Initial pH adjustment: Adjust the initial pH of the culture medium to 5.9 using 0.5 mol / L dilute hydrochloric acid or sodium hydroxide solution.

[0273] 5. Dispensing and sterilization: Dispense the prepared culture medium into culture containers, seal them, and place them in a high-pressure steam sterilizer for sterilization at 120°C for 25 minutes.

[0274] 6. Cooling and ready for use: After sterilization, slowly reduce the pressure and wait for the culture medium to cool naturally to 32°C before taking it out and placing it in a sterile clean environment for later use.

[0275] The compound acidic soil conditioner, by weight, comprises the following ingredients: 15 parts D5-N19 bacterial powder, 35 parts peat, 20 parts vermiculite powder, 20 parts corn cob powder, 18 parts wheat bran, 10 parts corn flour, 8 parts soybean meal powder, 6 parts potassium humate, 4 parts trehalose, 3 parts skim milk powder, 8 parts light calcium carbonate, 0.8 parts sodium carboxymethyl cellulose, and 2.5 parts fulvic acid.

[0276] A method for preparing a compound acidic soil conditioner includes the following steps:

[0277] 1. Carrier pretreatment: Peat, vermiculite powder, corn cob powder and wheat bran are crushed separately, passed through an 80-mesh standard sieve, mixed evenly according to the formula ratio, and placed in an autoclave at 121℃ for 25 minutes. After cooling to room temperature, they are ready for use.

[0278] 2. Preparation of nutrient matrix: Weigh corn flour, soybean meal, potassium humate, light calcium carbonate and fulvic acid according to the formula, mix them evenly, add an appropriate amount of purified water, stir until the moisture content is 33% and the mixture is moistened, sterilize at 118℃ for 20 minutes, and cool for later use.

[0279] 3. Preparation of protective agent solution: Weigh trehalose, skim milk powder and sodium carboxymethyl cellulose according to the formula, add sterile purified water and stir until completely dissolved to prepare a protective agent aqueous solution with a mass concentration of 12%. After filtration through a 0.22μm filter membrane for sterilization, it is ready for use.

[0280] 4. Adsorption and mixing of bacterial powder: D5-N19 bacterial powder and pretreated carrier are put into a horizontal mixer and stirred at low speed for 15 minutes to initially mix. Then, the nutrient matrix is ​​added to the mixer and stirred for another 15 minutes. Finally, the protective agent aqueous solution is sprayed and stirred for another 25 minutes until the material is uniform.

[0281] 5. Low-temperature drying: Spread the evenly mixed wet bacterial agent on a tray to a thickness of 1.6 cm, place it in a low-temperature ventilated drying oven, and dry it at 40°C until the moisture content of the material is 18%.

[0282] 6. Finished product packaging: Crush the dried inoculant, pass it through a 40-mesh sieve, package it quantitatively according to specifications, seal it, and store it in a cool, dry place.

[0283] Application: Apply compound acidic soil conditioner at 5% of the soil weight.

[0284] Example 4

[0285] The composite liquid culture medium and composite acidic soil conditioner of Example 1 were used and applied at 8% of the soil weight, with the other conditions being the same.

[0286] Example 5

[0287] The composite liquid culture medium and composite acidic soil conditioner of Example 1 were applied at 10% of the soil weight, with the other conditions remaining the same.

[0288] Comparative Example 1

[0289] D5-N19 cells were cultured in the original LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0) to prepare a liquid bacterial culture (OD). 600 =1.0), inoculated at 5% of soil weight.

[0290] Comparative Example 2

[0291] The D5-N19 mycelium powder from Example 1 was used, with peat as the carrier only (90 parts peat + 10 parts D5-N19 mycelium powder), without any other nutrients, protective or buffering components, at a dosage of 5%.

[0292] Comparative Example 3

[0293] The compound liquid culture medium was modified by removing betaine, and the remaining components and processes were exactly the same as in Example 1. After culturing the strain, the same improver as in Example 1 was prepared and applied at a rate of 5%.

[0294] Comparative Example 4

[0295] The compound acid soil conditioner removes light calcium carbonate and potassium humate, and the remaining components and processes are exactly the same as in Example 1, with an application rate of 5%.

[0296] Comparative Example 5

[0297] Apply quicklime at 0.3% of the soil weight (equivalent to neutralizing acidity), with all other conditions remaining the same.

[0298] Comparative Example 6

[0299] The LB liquid bacterial solution from Comparative Example 1 was used for inoculation at 10% of the soil weight, with all other conditions remaining the same.

[0300] Comparative Example 7

[0301] Commercially available Bacillus subtilis alkali-producing bacteria agent, viable count 10 9 CFU / g, applied at 5% of soil weight, with all other conditions remaining the same.

[0302] Verification using Examples 1-5 and Comparative Examples 1-7:

[0303] Basic experimental conditions:

[0304] The tested soil samples were collected from acidic red soil in Chongzuo, Guangxi Zhuang Autonomous Region. The soil was air-dried and passed through a 2mm sieve. The initial pH was 4.54, the initial exchangeable aluminum (A) was 12.3 mmol / kg, and the organic matter content was 2.46%. Each treatment pot weighed 1.5 kg, with three replicates. The soil was naturally incubated at room temperature for 60 days, with regular watering to maintain 60% of the field capacity. The A reduction rate was calculated based on the initial A content of the soil: A reduction rate = (initial A - 60-day A) / initial A × 100%; soil viable bacteria count was based on dry soil.

[0305] Experimental results (see Table 14) and comparative analysis:

[0306]

[0307] Conclusion Analysis:

[0308] 1. In all examples, the soil pH remained stable above 5.6 for 60 days, the exchangeable aluminum reduction rate was higher than 77%, and the soil viable bacteria count remained at 10. 5 -10 6 CFU / g achieved long-term stable improvement, with technical effects significantly superior to all comparative ratios.

[0309] 2. Comparative Example 1 used conventional LB medium. After 60 days, the soil pH dropped, and the number of viable bacteria was only 1.7% of that in Example 1. This proves that the composite medium of the present invention significantly improves the acid resistance and colonization ability of the strains, which is the core guarantee for the improvement effect.

[0310] 3. The improvement effects of Comparative Example 2 (single carrier) and Comparative Example 4 (without buffered complexing component) were significantly lower than those of Example 1, proving that the synergistic effect of carrier, nutrition, protection and buffer is the key to the long-term colonization and stable effect of the strain, and effectively alleviates the problem of pH drop in the later stage of single bacterial agent.

[0311] 4. In Comparative Example 6, the pH of the high-inoculation-volume liquid bacterial agent dropped significantly after 60 days, while the exchangeable aluminum rebounded severely. In contrast, in Example 5, the effect continued to improve under the same inoculation volume. This proves that the bacterial agent formulation of the present invention breaks the technical misconception that "high inoculation volume = good effect" and has achieved significant technical progress.

[0312] 5. Comparative Example 5: Quicklime showed rapid initial effects but declined significantly in the later stages, lacking sustained fertilization effects; Comparative Example 7: The improvement effect of commercially available microbial agents was far lower than that of this invention. This technology combines long-term effectiveness, comprehensiveness, and safety, demonstrating outstanding overall advantages.

Claims

1. An alkali-producing bacterium, characterized in that, The alkali-producing bacterium is *Providencia rettgeri*, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38612 and deposit date of May 9, 2026.

2. A method for isolating and screening alkali-producing bacteria as described in claim 1, characterized in that, Includes the following steps: (1) Samples were collected from alkaline soil and 10 samples were prepared. 4 -10 5 Series of diluted suspensions; (2) Spread the diluted suspension on NA medium plates and incubate at 28°C for 48 hours. Select single colonies for purification three times. (3) The purified single colony was inoculated into LB medium and cultured at 30°C for 48 hours. The pH value of the bacterial solution was measured to screen out potential alkali-producing bacteria that could increase the pH of the medium. (4) Potential alkali-producing bacteria were inoculated into a compound liquid culture medium and cultured at 30°C and 200r / min for 48 hours to screen out strains that could significantly increase the pH of the culture medium in an acidic environment.

3. The method for isolating and screening alkali-producing bacteria according to claim 2, characterized in that, The alkaline soil mentioned in step (1) was collected from the saline-alkali area of ​​Dongying, Shandong.

4. The method for isolating and screening alkali-producing bacteria according to claim 2, characterized in that, The composite liquid culture medium in step (3) includes the following raw materials: glucose, sucrose, soluble starch, tryptone, yeast extract powder, soybean meal powder, corn steep liquor powder, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, manganese sulfate, betaine, and sodium chloride.

5. The alkali-producing bacteria according to claim 2, characterized in that, The strain described in step (3) can increase the pH of the culture medium by more than 1.8 units when cultured in a compound liquid culture medium for 48 hours.

6. The application of alkali-producing bacteria screened by the isolation and screening method according to any one of claims 2-5 in the improvement of acidic soil.

7. The application of the alkali-producing bacteria according to claim 6 in the improvement of acidic soil, characterized in that, The inoculation amount of the alkali-producing bacteria is 5%-10% of the soil weight.

8. The application of the alkali-producing bacteria according to claim 7 in the improvement of acidic soil, characterized in that, The acidic soil is red soil, lateritic soil, or red soil with a pH value below 5.

5.

9. A compound acidic soil conditioner, characterized in that, It includes alkali-producing bacteria screened by the isolation and screening method as described in any one of claims 2-5 and agriculturally acceptable vectors.

10. The composite acidic soil conditioner according to claim 9, characterized in that, The product comprises the following ingredients in parts by weight: alkali-producing bacteria powder, peat, vermiculite powder, corn cob powder, wheat bran, corn flour, soybean meal powder, potassium humate, trehalose, skim milk powder, light calcium carbonate, sodium carboxymethyl cellulose, and fulvic acid.

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