Cold-tolerant bacterium, microbial inoculant, and preparation method and application thereof

Abstract

This invention provides a strain of psychrophilic bacillus, a microbial inoculum, its preparation method, and its application, relating to the field of microbial geochemistry. The psychrophilic bacillus is a strain of psychrophilic bacillus (…). Psychrobacter SL-1, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34780, is a psychrophilic bacillus provided in this invention. It exhibits excellent low-temperature adaptability, successfully overcoming the limitation of conventional microorganisms in high-altitude, extreme environments. When applied to barren environments, it significantly increases the content of available phosphorus, available potassium, alkaline nitrogen, and organic carbon in the substrate, thoroughly improving the nutrient-deficient state of the substrate and providing a highly efficient biological engine for the ecological restoration of degraded soils and tailings at high altitudes.

Description

Technical Field

[0001] This invention relates to the field of microbial geochemistry, and more specifically, to a strain of psychrophilic bacillus, a microbial agent, its preparation method, and its application. Background Technology

[0002] Soil ecological restoration is a key technical means to improve degraded environments and restore land productivity. In extreme geographical areas such as high-altitude regions with fragile ecological backgrounds, soils generally face severe challenges such as persistently low temperatures and poor substrate. At the same time, localized industrial mining activities further exacerbate soil degradation and micro-ecological imbalances, posing significant challenges to conventional ecological restoration work.

[0003] For soil remediation in such extreme environments, microbial remediation technology has made some progress, with related research mainly focusing on screening functional strains that can adapt to low-temperature environments. Existing technologies have isolated and identified functional microorganisms including Bacillus, Pseudomonas, and Burkholderia, with the aim of utilizing them to achieve soil improvement at low temperatures.

[0004] However, existing technologies still have significant shortcomings in practical applications. First, the activity and reproductive capacity of most conventionally screened phosphorus- and potassium-solubilizing microorganisms are severely inhibited when faced with real extreme low-temperature environments. Second, extremely infertile soil substrates cannot provide the nutritional support required for the initial colonization of exogenously introduced microorganisms, resulting in low survival rates of the strains. Furthermore, special microbial resources with natural advantages in adapting to extreme low temperatures have not been fully explored and effectively transformed, leading to a serious lack of diversity and environmental specificity in existing soil amendment agents for high-altitude areas.

[0005] In summary, the existing technological system urgently lacks specialized microbial resources that can highly adapt to and stably survive in high-altitude and barren environments. How to effectively overcome the difficulties in colonization and low activity of exogenous microorganisms in extreme habitats, and to substantially increase the content of key nutrients in degraded soils, is a pressing technical challenge that needs to be addressed in this field.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a strain of psychrophilic bacillus, a microbial inoculant, and a method for preparing the microbial inoculant. The psychrophilic bacillus SL-1 can maintain high activity in extreme cold environments and fundamentally solves the problem of ecological restoration of degraded substrates at high altitudes by significantly increasing the content of available phosphorus, available potassium, alkaline nitrogen and organic carbon in barren soil or tailings.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a strain of psychrobacter, namely Psychrobacter SL-1, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 34780.

[0009] Secondly, the present invention provides a microbial inoculant containing psychrophilic bacilli as described in the foregoing embodiments.

[0010] In an optional embodiment, the microbial agent is a liquid agent or a solid agent.

[0011] In an optional embodiment, when the microbial agent is a liquid agent, the liquid agent is a bacterial suspension obtained by fermenting and culturing the psychrophilic bacillus at a temperature of 4°C to 18°C; and / or, When the microbial agent is a solid agent, the solid agent includes a carrier and the psychrophilic bacillus loaded on the carrier.

[0012] In an optional embodiment, the carrier is biochar.

[0013] In an optional embodiment, the solid bacterial agent is prepared by cross-linking and immobilizing a suspension of the psychrophilic bacillus with biochar, followed by encapsulation with sodium alginate and calcium chloride; and / or, The biochar is cow dung-derived biochar and / or straw-derived biochar; and / or, The biochar is produced by pyrolysis of cow dung or straw at 400℃ to 450℃.

[0014] Thirdly, the present invention provides a method for preparing a microbial inoculant as described in the foregoing embodiments, comprising: Provide a bacterial suspension of the aforementioned psychrophilic bacillus; The bacterial suspension is mixed with the biochar and subjected to adsorption treatment to obtain an adsorption mixture; The adsorption mixture was gelled and immobilized using sodium alginate solution and calcium chloride solution to obtain the solid bacterial agent.

[0015] In an optional embodiment, the ratio (v / w) of the bacterial suspension to the biochar is 1:(5-20); and / or, The sodium alginate solution has a mass fraction of 2%; and / or, The calcium chloride solution has a mass fraction of 4%; and / or, The gelation and embedding fixation process includes: uniformly mixing the adsorbed mixture with a 2% (w / v) sterile sodium alginate solution at a volume ratio of 2:1 to 1:1.5 to obtain a mixed colloidal solution; crosslinking the mixed colloidal solution using a 4% (w / v) calcium chloride solution; and / or, The bacterial suspension was obtained by fermentation culture of the psychrophilic bacillus at a temperature of 4°C to 18°C.

[0016] Fourthly, the present invention also provides the application of psychrophilic bacilli as described in the foregoing embodiments, or microbial agents as described in the foregoing embodiments, in soil improvement or tailings ecological restoration.

[0017] In an optional implementation, the soil improvement or tailings ecological restoration environment is an area with an altitude of not less than 1500m; and / or, The application includes methods for increasing the content of target elements in soil or tailings; wherein the target elements include at least one of available phosphorus, available potassium, alkaline nitrogen, and organic carbon; and / or, When applying the liquid microbial agent, the application rate is 10 mL / kg to 30 mL / kg of the target substrate; and / or, When applying the solid microbial agent, the application rate is 10 g / kg of the target substrate; the target substrate is soil or tailings.

[0018] Compared with the prior art, the psychrophilic bacillus ( ) provided by the present invention Psychrobacter SL-1, a special microbial resource naturally adapted to extreme high-altitude and cold environments, possesses excellent low-temperature adaptability. Unlike conventional phosphorus- and potassium-solubilizing microorganisms whose metabolic activity and reproductive capacity are severely inhibited under low-temperature conditions, this specific strain can maintain stable physiological metabolic activity and vigorous growth in harsh low-temperature ecological environments. This characteristic effectively fills the gap in the current field of high-altitude ecological restoration, which lacks specific low-temperature microbial resources, and directly overcomes the fundamental deficiency that conventional exogenous microorganisms are difficult to survive and colonize in extreme habitats.

[0019] Meanwhile, this psychrophilic bacillus exhibits remarkably advanced biogeochemical transformation capabilities. When directly applied to harsh substrates such as high-altitude, infertile soils or tailings sands, this strain, through its highly efficient metabolic activity at low temperatures, significantly activates and increases the content of the four core nutrients in the substrate: available phosphorus, available potassium, alkaline nitrogen, and organic carbon. This highly efficient element activation ability fundamentally improves the long-term nutrient deficiency and infertility of degraded substrates, effectively activating the inherent nutrient cycling mechanism within the target area.

[0020] The stable survival and efficient elemental transformation output of this strain in fragile ecosystems provide an extremely reliable and highly active biological engine for the reconstruction of the physicochemical structure of degraded soils at high altitudes and the ecological restoration of tailings in mining areas, providing strong biological support for improving the extremely fragile ecological base of extreme geographical areas from the root. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 This is a colony morphology diagram of psychrophilic bacillus SL-1 in Example 1 of this application; Figure 2 This is a phylogenetic tree of psychrophilic bacillus SL-1 constructed based on the 16S rDNA sequence in Example 1 of this application; Figure 3 This is a graph showing the changes in available phosphorus and available potassium content of psychrophilic bacillus SL-1 in Example 1 of this application after 5 days of fermentation in liquid fermentation medium; Figure 4 The images shown are scanning electron microscope (SEM) images of the solid bacterial agent in Example 3 of this application (wherein, Image A is a 10,000x magnified image of the bacterial cell attachment state, and Image B is a 1,000x magnified image of the carrier spatial structure). Figure 5 This is a diagram showing the relative abundance distribution of microbial communities at the phylum level in the tailings substrate of each treatment group in Example 4 of this application; Figure 6 This is a diagram showing the relative abundance distribution of microbial communities at the genus level in the tailings matrix of each treatment group in Example 4 of this application.

[0023] Preservation information: Accession number: CGMCC No. 34780; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: June 6, 2025; Sample name: Psychrophilic bacilli (Psychrophilic bacilli) Psychrobacter SL-1. Detailed Implementation

[0024] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0025] This application provides a psychrobacter strain with excellent low-temperature adaptability and nutrient activation ability. The specific classification of the psychrobacter is *Psychrobacter*, and its strain code is SL-1. This psychrobacter SL-1 has been deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on June 6, 2025, with accession number CGMCC No. 34780.

[0026] This psychrophilic bacillus ( Psychrobacter SL-1 is a typical low-temperature adapted microorganism, naturally adapted to extreme ecological environments such as high altitudes and cold regions, and possesses a significant advantage in stably carrying out physiological metabolism and vigorous growth under low-temperature stress. This strain has excellent biogeochemical transformation capabilities, enabling it to efficiently colonize in barren substrates through its unique metabolic activities, and continuously convert nutrients in the substrate that are difficult to use directly into available forms.

[0027] Specifically, the psychrophilic bacillus ( Psychrobacter When applied to target substrates such as degraded soils or tailings sands, SL-1 can significantly and synergistically increase the content of available phosphorus, available potassium, alkaline nitrogen, and organic carbon in the substrate. This strain effectively overcomes the deficiency of conventional phosphorus- and potassium-solubilizing microorganisms in low-temperature environments. By directly improving the nutrient-deficient state of the bottom substrate in extreme geographical habitats, it provides a highly active key biological resource for soil improvement, degraded habitat reconstruction, and ecological restoration of tailings ponds in high-altitude areas.

[0028] This application also provides a microbial agent containing a core active ingredient: Psychrobacter SL-1, which has the accession number CGMCC No. 34780.

[0029] The microbial agent is a biological preparation based on the psychrophilic bacillus SL-1, which can be practically spread and applied. Due to the natural low-temperature adaptation advantage and excellent biogeochemical transformation function of the psychrophilic bacillus SL-1, the live strain contained in the microbial agent can be effectively released and rapidly colonized in the target substrate after being applied to the external environment.

[0030] In practical applications, the microbial agent not only maintains the physiological activity of psychrophilic bacillus SL-1 during storage and transportation, but also ensures a sufficient initial viable count when added to degraded substrates (such as barren soils or tailings sand in high-altitude and cold regions). After the agent enters the target habitat, psychrophilic bacillus SL-1, through its vigorous metabolic activity, breaks the binding of nutrients by minerals, thereby significantly and stably increasing the content of available phosphorus, available potassium, alkaline nitrogen, and organic carbon in the substrate within the application area.

[0031] Furthermore, to adapt to the ecological restoration needs of different scales and working conditions, the microbial agent can be processed into different dosage forms according to conventional preparation processes. For example, the microbial agent can be a liquid agent obtained directly through fermentation culture; or, to further improve the strain's stress resistance and extend its shelf life, the microbial agent can also be a solid agent prepared through specific carrier loading and immobilization processes. Regardless of the dosage form used, the survival rate and metabolic activities such as phosphorus solubilization and potassium solubilization of the psychrophilic bacillus SL-1 must be maintained.

[0032] In some embodiments, the microbial agents provided in this application can be prepared in liquid or solid form to adapt to different scales and severity of ecological restoration conditions.

[0033] When the microbial agent is a liquid agent, it typically contains live psychrophilic bacilli (…). Psychrobacter This liquid microbial agent is a fluid suspension of SL-1 cells and an aqueous medium. It features a simple preparation process, a high initial effective viable cell count, and is rich in active substances produced during bacterial metabolism (such as free enzymes and organic acids). In practical applications, the liquid microbial agent can directly and rapidly penetrate into the soil pores of the target area through spraying or other methods, allowing the bacterial strains to quickly contact the substrate and exert nutrient activation effects. It is suitable for scenarios requiring the rapid establishment of a dominant microbial community.

[0034] When the microbial agent is a solid agent, it is composed of the psychrophilic bacillus (… Psychrobacter SL-1 cells are formed by binding to a solid carrier structure. The solid form provides a physical buffer barrier (miniature shelter) for the fragile living microbial cells, effectively resisting direct physical or chemical shocks from harsh external environments such as extreme low temperatures, drought, and extremely poor substrates at high altitudes. Solid microbial agents are not only easier to package, transport over long distances, and store for extended periods, but their inherent structural characteristics, when applied to degraded soil or tailings sand, help psychrophilic bacteria achieve efficient initial colonization in poor substrates and create a slow-release effect, thus ensuring the microbial agent exerts a lasting and long-lasting bio-improvement effect in extreme ecological environments.

[0035] In order to further adapt to ecological restoration conditions of varying severity and maximize the biological activity of the core strains, the microbial agents provided in this application have a variety of preferred formulations and preparation methods.

[0036] In a preferred embodiment, when the microbial agent is a liquid agent, the liquid agent is specifically the psychrophilic bacillus (…). Psychrobacter The live bacterial suspension obtained by fermenting SL-1 at temperatures ranging from 4℃ to 18℃ (e.g., 4℃, 5℃, 7℃, 9℃, 11℃, 13℃, 15℃, 16℃, 17℃, 18℃, etc.). Since this strain is a typical low-temperature adapted microorganism, strictly controlling the fermentation temperature within the range of 4℃ to 18℃ can highly simulate its natural high-altitude in-situ habitat, avoiding the destruction of cold-adapted enzyme systems by high temperatures. The bacterial suspension obtained under these temperature parameters not only has an extremely high concentration of live cells, but also contains a large amount of extracellular metabolites (such as phospholytic enzymes and organic acids) secreted by this strain under cold stress. When this liquid bacterial agent is directly applied to the target area, the free bacterial cells and their metabolites can rapidly diffuse into the soil matrix pores, quickly initiating the activation process of environmental nutrients.

[0037] It should be noted that the psychrophilic bacillus SL-1 described in this embodiment exhibits significant psychrophilic / cold-resistant characteristics, maintaining good growth and metabolic activity across a wide temperature range of 4℃ to 18℃. Although its growth rate and metabolite accumulation reach an optimal balance at around 10℃, the strain can still maintain effective proliferation through its own cold-resistance mechanism at a low temperature of 4℃, which is close to the extreme end. While its metabolic activity is enhanced at temperatures close to 18℃, it still maintains good bacterial community stability, thereby ensuring that the prepared bacterial suspension has a stable number of viable bacteria.

[0038] In another preferred embodiment, when the microbial agent is a solid agent, the solid agent includes a carrier having a certain three-dimensional physical structure, and the psychrophilic bacillus loaded on the carrier by physical or chemical means. PsychrobacterSL-1. In extreme high-altitude, frigid, and extremely barren tailings substrates, free-living microorganisms are highly susceptible to severe environmental shocks and lose their activity. By introducing a physical carrier, a microscopic shelter space is provided for the psychrophilic bacilli to buffer against external stresses. The carrier, with its unique structural properties (such as porosity), can not only firmly attach to or adsorb the bacteria but also retain moisture and basic nutrients in the local microenvironment, thus forming a synergistic protective system with the psychrophilic bacilli loaded on it. After being applied to degraded habitats, this solid microbial agent can significantly resist the impact of harsh factors such as low temperatures and heavy metals, maintain the initial viable bacterial count, and achieve slow-release and long-term colonization of psychrophilic microorganisms in barren substrates, ensuring the sustainability of ecological improvement effects.

[0039] In some embodiments, the carrier is biochar. That is, the microbial agent is a solid agent, and the solid agent uses biochar as a physical carrier.

[0040] In some embodiments, the solid bacterial agent is formed by immobilizing the suspension of the psychrophilic bacillus by the biochar, followed by cross-linking and embedding with sodium alginate and calcium chloride.

[0041] More preferably, the solid bacterial agent has a dual composite structure of "internal support-external embedding". It is formed by first fully adsorbing the bacterial suspension of the Psychrobacter SL-1 by the biochar, and then embedding and fixing it by chemical cross-linking reaction between sodium alginate solution and calcium chloride solution.

[0042] Specifically, in this dual structure, biochar, with its well-developed porous structure and large specific surface area, provides a physical microenvironment (internal support) that protects free psychrophilic bacteria cells from direct impacts from the external environment, and can retain moisture and nutrients within its pores. Meanwhile, the calcium alginate three-dimensional network gel macromolecules formed by the cross-linking of sodium alginate and calcium chloride (external encapsulation) form a tough, semi-permeable protective membrane on the outside of the biochar-bacterial complex. This gel membrane allows the free transport of oxygen and nutrient ions to maintain bacterial metabolism, while effectively resisting physical damage from high-altitude, low-temperature ice crystals and the toxicity of heavy metals from tailings. The synergistic effect of these two components not only greatly improves the survival rate of psychrophilic bacteria in barren and harsh substrates, but also endows the bacterial agent with excellent sustained-release properties, enabling the live bacteria to exert their phosphorus-solubilizing, potassium-solubilizing, and carbon-nitrogen cycling functions in the habitat for a long and sustained period.

[0043] In some embodiments, the biochar is cow dung biochar and / or straw biochar.

[0044] In some embodiments, the biochar is obtained by pyrolysis of cow dung or straw at 400°C to 450°C. For example, the temperatures can be 400°C, 405°C, 410°C, 415°C, 420°C, 425°C, 430°C, 435°C, 440°C, 450°C, etc.

[0045] In order to balance ecological benefits and carrier performance, the biochar is cow manure-derived biochar or straw-derived biochar; and the biochar is obtained by pyrolysis of cow manure or straw under medium and low temperature conditions of 400℃ to 450℃.

[0046] Using cow dung or straw as precursors not only achieves the resource-based recycling of agricultural waste, but also allows these two natural biomass to form optimal physicochemical configurations through pyrolysis within a specific temperature window of 400℃ to 450℃. This temperature range avoids excessive graphitization and extreme hydrophobicity caused by high-temperature pyrolysis, ensuring that the surface of the resulting biochar retains abundant oxygen-containing functional groups (such as hydroxyl and carboxyl groups). This not only greatly enhances its adsorption affinity for hydrophilic bacterial suspensions and water-soluble nutrients in the environment, but also provides basic nutritional support for the early colonization and reproduction of psychrophilic bacteria from the residual nutrients in cow dung and straw.

[0047] This application provides a method for preparing a microbial agent. This method, through a multi-stage immobilization process, aims to prepare a solid composite microbial agent with extremely high environmental adaptability and long-lasting sustained-release function. The preparation method includes: Step S1: Provide a bacterial suspension of the psychrophilic bacillus.

[0048] In this step, the psychrophilic bacillus ( Psychrobacter SL-1 was inoculated into a suitable liquid culture medium for propagation. During the culture process, the bacteria proliferated rapidly and dispersed evenly in the liquid medium, forming a highly active free bacterial suspension. This suspension is not only rich in high concentrations of live microbial cells, but also contains physiologically active extracellular products secreted by the bacteria during metabolism, providing core bioactive components for subsequent processes.

[0049] Step S2: Mix the bacterial suspension with the biochar and perform adsorption treatment to obtain an adsorption mixture.

[0050] The bacterial suspension obtained above was thoroughly contacted and mixed with porous biochar in a certain proportion. During this process, due to the large specific surface area and rich microporous network of biochar, coupled with the polar functional groups typically present on its surface, the bacterial suspension rapidly penetrated deep into the pores of the biochar under the combined influence of capillary forces, physical van der Waals forces, and surface charge attraction. Free psychrophilic bacilli cells, along with water and nutrients from the fermentation broth, were firmly adsorbed and retained within the micropores of the biochar, thus completing the first stage of physical fixation and obtaining an adsorbed mixture. The pores of the biochar here act as a miniature habitat for microorganisms, providing them with initial protection and a nutrient reservoir.

[0051] Step S3: The adsorbent mixture is gelled and embedded using sodium alginate solution and calcium chloride solution to obtain the solid bacterial agent.

[0052] The aforementioned gelation embedding and fixation technology refers to the technique of using natural or synthetic polymer materials to undergo phase change (from liquid cross-linking and solidification into a three-dimensional network gel) under specific conditions to encapsulate and confine target substances or cells within their network framework structure.

[0053] Specifically, the adsorbed mixture obtained in step two is dispersed in a sodium alginate aqueous solution of a certain concentration and mixed evenly. Subsequently, this mixture is reacted with a calcium chloride solution. At this time, the sodium ions on the long-chain macromolecules of sodium alginate are rapidly replaced by divalent calcium ions provided by calcium chloride. The divalent calcium ions undergo multiple coordination bonding with the carboxyl groups on the adjacent alginate macromolecule chains, instantly constructing a tight three-dimensional "egg-box" three-dimensional network cross-linked structure, thus forming a water-insoluble calcium alginate hydrogel.

[0054] Through the aforementioned cross-linking reaction, biochar particles carrying psychrophilic bacteria are densely encapsulated within a calcium alginate gel network, forming the final solid bacterial agent. This gel shell constructs a highly efficient semi-permeable physical barrier, allowing not only the free permeation and exchange of oxygen and water-soluble small molecule nutrients essential for bacterial life activities, but also effectively preventing mechanical damage from ice crystals caused by extreme external temperatures, as well as the acute toxicity of heavy metal ions in harsh environments such as tailings. This multi-stage process organically combines "biochar internal support adsorption" with "gel shell encapsulation," exhibiting a significant synergistic effect and greatly enhancing the survival rate and colonization capacity of exogenous microorganisms in barren, high-altitude, and extreme environments.

[0055] In some embodiments, the ratio (v / w) of the bacterial suspension to the biochar is 1:(5-20).

[0056] It should be noted that the choice of mixing ratio mainly depends on the adsorption capacity of the carrier and the required viable bacteria concentration in the final product. Within a ratio range of 1:5 to 1:20, the carrier can effectively load the bacterial suspension. Lower ratios (e.g., 1:5) are suitable for scenarios requiring high initial viable bacteria concentrations, while higher ratios (e.g., 1:20) are beneficial for achieving a longer-lasting, slow release through the protective effect of the carrier. Through extensive experimentation, the inventors discovered that a ratio of approximately 1:10 provides the most balanced balance between the particle size distribution and loading efficiency of the bacterial agent.

[0057] In some embodiments, the sodium alginate solution has a mass fraction of 2%.

[0058] In some embodiments, the calcium chloride solution has a mass fraction of 4%.

[0059] In some embodiments, the gelation embedding and fixation includes: uniformly mixing the adsorbed mixture with a 2% (w / v) sterile sodium alginate solution at a volume ratio of 2:1 to 1:1.5 to obtain a mixed colloidal solution; and crosslinking the mixed colloidal solution with a 4% (w / v) calcium chloride solution.

[0060] In some embodiments, the bacterial suspension is obtained by fermentation culture of the psychrophilic bacillus at a temperature of 4°C to 18°C.

[0061] It should be noted that psychrobacters are a class of microorganisms capable of maintaining vigorous metabolic activity in low-temperature environments. Fermentation within this temperature range aims to conform to the physiologically optimal growth curve of this strain, maintaining suitable cell membrane fluidity and maximizing the activity of its cold-adapted enzyme systems, thereby obtaining a high-concentration bacterial suspension in a highly physiologically active state. The fermentation temperature can be, for example, 4℃, 5℃, 7℃, 9℃, 11℃, 13℃, 15℃, 16℃, 17℃, 18℃, etc.

[0062] After obtaining the bacterial suspension, it is mixed with biochar as a carrier for adsorption. To ensure that water and bacteria can fully penetrate the rich pores of the biochar, while avoiding waste of free bacteria or idle carrier space, the ratio (v / w) of the bacterial suspension to the biochar is strictly controlled between 1:(5-20). This ratio parameter can establish an optimal solid-liquid microenvironment, allowing bacteria to firmly adhere to the carrier under the action of capillary forces. For example, the ratio (v / w) of the bacterial suspension to the biochar can be 1mL:5g, 1mL:6g, 1mL:8g, 1mL:10g, 1mL:12g, 1mL:14g, 1mL:15g, 1mL:17g, 1mL:19g, 1mL:20g, etc. After thorough stirring or impregnation, an adsorption mixture loaded with microorganisms is obtained. To further improve the sustained-release effect and stress resistance of the microbial agent after application, the above-mentioned adsorbent mixture was gelled and embedded. The specific process is as follows: the adsorbent mixture was uniformly mixed with a 2% (w / v) sterile sodium alginate solution, wherein the volume ratio of the adsorbent mixture to the sterile sodium alginate solution was controlled between 2:1 and 1:1.5, resulting in a homogeneous mixed colloidal solution. For example, the volume ratio can be 2 mL:1 mL, 1.8 mL:1 mL, 1.5 mL:1 mL, 1.2 mL:1 mL, 1 mL:1 mL, 1 mL:1.1 mL, 1 mL:1.2 mL, 1 mL:1.3 mL, 1 mL:1.4 mL, 1 mL:1.5 mL, etc. A 2% (w / v) sodium alginate solution was chosen because this concentration combines good operable rheological properties with a suitable pore size after solidification, facilitating mixing and extrusion while allowing nutrients and metabolic waste to diffuse freely within the formed gel network.

[0063] Subsequently, the mixed colloidal solution was cross-linked using a 4% (w / w) calcium chloride solution. Specifically, the mixed colloidal solution was added dropwise to the 4% calcium chloride solution at a uniform rate. Upon contact with calcium ions, sodium alginate rapidly undergoes ion exchange and cross-linking polymerization, forming a three-dimensional gel network with an "egg-box" structure, firmly encapsulating the biochar containing psychrophilic bacteria at its core. The 4% calcium chloride concentration ensures rapid molding and sufficient mechanical strength for the microspheres, while controlling the osmotic pressure within a safe range to prevent bacterial dehydration and inactivation.

[0064] The gelation and embedding immobilization technology used in this scheme (i.e., using biochar as the core for adsorption and colonization, and an outer polymer hydrogel as a protective barrier) constructs a micro-bioreactor with a "dual protection" mechanism at the micro level, which can effectively buffer extreme pH, heavy metal toxicity and other environmental stresses, and significantly extend the action cycle and shelf life of the microbial agent.

[0065] This application also provides the application of psychrophilic bacilli as described in the foregoing embodiments, or microbial agents as described in the foregoing embodiments, in soil improvement or tailings ecological restoration.

[0066] Degraded soils and tailings typically face ecological problems such as structural compaction, lack of organic matter, extremely low levels of available nutrients, and potential heavy metal toxicity. Applying the psychrophilic bacillus or microbial agents provided in this invention to the target substrate (soil or tailings) allows the strain to utilize its efficient metabolic activity to secrete organic acids, extracellular enzymes, and high-molecular polymers, thereby accelerating mineral weathering, improving the physicochemical properties of the substrate, and establishing a favorable micro-ecological environment for subsequent plant colonization.

[0067] In some embodiments, the environment for soil improvement or tailings ecological restoration is an area with an altitude of not less than 1500m.

[0068] High-altitude regions experience consistently low temperatures, which severely inhibits the activity of conventional microorganisms, hindering their ecological restoration efforts. However, the psychrophilic bacilli used in this invention possess excellent cold adaptation mechanisms, maintaining cell membrane fluidity and enzyme activity under low-temperature stress, thus ensuring the effectiveness of ecological restoration projects in high-altitude and cold regions. Regarding the altitude range of "not less than 1500m," in practical engineering applications, the altitude of the restoration environment can be, for example, 1500m, 1800m, 2000m, 2500m, 3000m, 3500m, 4000m, 4500m, 4800m, 5000m, etc.

[0069] In some embodiments, the application includes increasing the content of a target element in soil or tailings; wherein the target element includes at least one of available phosphorus, available potassium, available nitrogen, and organic carbon.

[0070] After colonizing the target substrate, psychrophilic bacteria transform nutrients that were originally fixed by the mineral lattice or in a slow-release state into readily available forms (such as available phosphorus and available potassium) that can be directly absorbed and utilized by plant roots through a series of biochemical reactions, including biological nitrogen fixation, phosphorus solubilization, potassium solubilization, and decomposition of organic macromolecules. This significantly increases the level of alkaline nitrogen and organic carbon reserves in the substrate, fundamentally solving the problem of extremely poor soil quality in tailings or degraded soils.

[0071] In some embodiments, when the liquid microbial agent is applied, the dosage is 10 mL / kg to 30 mL / kg of the target substrate. Preferably, the dosage is 20 mL / kg.

[0072] In some embodiments, when the solid microbial agent in the microbial agent is applied, the application rate is 10 g / kg of the target substrate; the target substrate is soil or tailings.

[0073] To ensure the rapid formation of a dominant microbial population in the target substrate and achieve optimal improvement results, while also considering economic costs, this invention optimizes the application rate of the microbial agent. Specifically, when using the liquid microbial agent for remediation, the standard application rate is controlled at 10 mL / kg to 30 mL / kg of target substrate; when using the solid microbial agent, the standard application rate is controlled at 10 g / kg of target substrate. In actual construction operations, spraying, mechanical rotary tillage mixing, or furrow application can be used to ensure sufficient and uniform contact between the microbial agent and the target substrate (i.e., the soil to be improved or the tailings to be remediated), thereby initiating the in-situ ecological remediation process.

[0074] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0075] Example 1: Screening and identification of psychrophilic bacillus SL-1 This embodiment provides a psychrophilic bacillus with excellent low-temperature adaptability and phosphorus solubilization, potassium solubilization, and nitrogen fixation functions. Psychrobacter The process of screening, isolation, purification and molecular biological identification of SL-1.

[0076] 1. Experimental Method: (1) Sample collection and processing: Soil samples were collected from tailings ponds at an altitude of 1500m or above. The top 0-20cm soil was selected, and after removing impurities such as stones and plant debris, it was placed in a sterile sealed bag, stored at 4℃ and quickly brought back to the laboratory. The strain screening was completed within 24 hours.

[0077] (2) Culture medium preparation: (1) Low-temperature enrichment medium (g / L): 10g glucose, 2g yeast extract, 5g calcium phosphate, 8g potassium feldspar powder, 3g sodium chloride, 0.5g magnesium sulfate, 1000mL distilled water, pH adjusted to 7.0-7.2. Autoclave at 121℃ for 25min, cool to 4℃~18℃ for later use. Suitable for low-temperature enrichment culture of psychrophilic bacteria.

[0078] (2) Low-temperature separation medium (g / L): Add 20g of agar powder to the above enrichment medium, and make solid plates with the same other components and sterilization conditions.

[0079] (3) Low-temperature fermentation medium: LB liquid medium was autoclaved at 121℃ for 25 min and cooled to 4℃~18℃ for later use; Alexandrov medium: sucrose 5.0g, calcium carbonate 0.1g, magnesium sulfate 15.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.2g, ferric chloride 0.5g, soil minerals 2~5g. Cool to 4℃~18℃ for later use.

[0080] (4) Strain screening: 1) Weigh 10g of soil sample, add 90mL of sterile physiological saline, and place in a shaker at 4℃ and 120r / min for 30min to prepare 10 -1 Soil suspension, serially diluted to 10 -6 10 -7 ; 2) Take 10 respectively -6 10 -7 Dilute 0.2 mL, spread on a low-temperature separation medium plate, and incubate upside down in a 10℃ constant temperature incubator for 72 h; 3) Single colonies with regular morphology and large clear zones (phosphorus and potassium solubilizing zones) were selected from the plates. After secondary screening and verification, the clear zone formed by strain SL-1 on Alexandrov medium plates had clear edges, and the ratio of its diameter (D) to colony diameter (d) was significantly better than that of conventional potassium-solubilizing bacteria, preliminarily confirming its efficient ability to activate inorganic phosphorus and potassium. Combined with the acid production experiments (methyl red experiment and sugar fermentation experiment) results in Table 1, SL-1 can produce organic acids through metabolism, thereby achieving chemical chelation and physical degradation of insoluble phosphorus and potassium in the substrate. Purification was performed using the streak plating method, repeated three times, to obtain a pure strain, designated SL-1.

[0081] 2. Experimental Results and Analysis: (1) Morphological identification results: refer to Figure 1 SL-1 was inoculated into a low-temperature isolation medium and cultured at 10°C for 48 hours. The colony characteristics were observed: the colonies were round, smooth, with neat edges, milky white, and opaque, with a diameter of about 1-2 mm. After Gram staining and microscopic examination, the bacteria were Gram-negative short rods, without spores, and existed singly or in pairs.

[0082] In addition to the morphological characteristics mentioned above, this application has quantitatively / semi-quantitatively verified the phosphorus solubilization, potassium solubilization, and nitrogen fixation capabilities of psychrophilic bacillus SL-1.

[0083] The phosphorus-solubilizing and potassium-solubilizing activities were determined using the clear zone method: SL-1 was inoculated onto Monkina solid medium (phosphorus-solubilizing) and Alexandrov solid medium (potassium-solubilizing) plates and incubated at 10℃ for 72 h. The results showed that distinct clear zones formed around the colonies. Calculations showed that the ratio of the phosphorus-solubilizing zone diameter D to the colony diameter d (D / d) was 2.3, and the ratio of the potassium-solubilizing zone diameter D to the colony diameter d (D / d) was 1.9.

[0084] Meanwhile, the nutrient activation capacity was quantitatively determined using a liquid shake-flask experiment: SL-1 was inoculated into the corresponding liquid culture medium and cultured at 10℃ and 120 r / min for 5 days with shaking. The effective phosphorus concentration in the fermentation broth was found to be 281.57 mg / L higher than that of the uninoculated control group, and the available potassium concentration was 35.22 mg / L higher (reference). Figure 3 Furthermore, the strain grew well in nitrogen-free medium, confirming that it possesses significant synergistic functions of low-temperature phosphorus solubilization, potassium solubilization, and nitrogen fixation.

[0085] (2) Detection of physiological and biochemical characteristics: The physiological and biochemical characteristics of the psychrophilic bacillus SL-1 were detected, and the results are shown in Table 1: Simultaneously, physiological and biochemical experiments were conducted, and the results are as follows: Table 1. Results of physiological and biochemical characteristics experiments

[0086] As shown in Table 1 ("+" indicates positive and "-" indicates negative), this strain has the ability to utilize multiple carbon sources and has an active nitrogen metabolism pathway, which lays the physiological foundation for its ability to secrete organic acids and activate mineral nutrients through metabolism in the extremely barren environment at high altitudes.

[0087] (3) Molecular biological identification results: Genomic DNA was extracted from strain SL-1 and amplified by PCR using universal primers for bacterial 16S rDNA. This included: 27F: (SEQ ID NO. 2) 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: (SEQ ID NO. 3) 5'-GGTTACCTTGTTACGACTT-3'.

[0088] PCR reaction system (50μL): 10×PCR Buffer 5μL, dNTP Mixture (2.5mmol / L) 4μL, forward and reverse primers 1μL each, template DNA 2μL, Taq DNA polymerase 0.5μL, sterile distilled water 36.5μL; PCR reaction procedure: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 10 min, store at 4℃. The PCR amplification product was purified and sequenced, and its 16S rDNA sequence was obtained as shown in SEQ ID NO. 1. Specifically:

[0089] The sequencing results were compared with known sequences in the NCBI database for homology, and a phylogenetic tree was constructed (see reference). Figure 2 The results showed that SL-1 was associated with the genus *Psychrophilus* (…). Psychrobacter With a homology of over 99%, the strain was identified as a psychrophilic bacillus.

[0090] The psychrophilic bacillus SL-1 was deposited on June 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34780.

[0091] Example 2: Preparation of Psychrophilic Bacillus SL-1 liquid inoculum (fermentation broth) This embodiment provides a specific method for preparing a high-concentration liquid fermentation agent using the above-mentioned psychrophilic bacillus SL-1, in order to examine the propagation ability of this strain under low-temperature conditions.

[0092] 1. Experimental Method: (1) Preparation of seed liquid: Preserved psychrophilic bacteria ( Psychrobacter sp. Strain strain SL-1 was aseptically inoculated into Erlenmeyer flasks containing low-temperature fermentation medium. The culture was incubated at 4–18°C and 120 rpm for 18 h to obtain a seed culture in the logarithmic growth phase. The absorbance (OD) of the culture medium at 600 nm was monitored using a spectrophotometer. 600 The concentration of the seed solution should be controlled within the range of 1.0 to 1.5.

[0093] (2) Expanding fermentation and microbial agent production: Under aseptic conditions, the above seed culture was transferred to a fermenter containing fresh, low-temperature enrichment medium at an inoculation rate of 2% (v / v). The culture was then incubated with shaking at 10°C and 120 rpm for 20 hours to obtain a high-density bacterial suspension of psychrophilic bacillus SL-1, which is the liquid inoculum. The temperature throughout the fermentation process was strictly controlled within the range of 4–18°C.

[0094] 2. Experimental Results: As determined by plate count, the viable bacterial concentration of the liquid bacterial agent obtained in this embodiment is not less than 1×10⁻⁶. 9 The CFU / mL value indicates that this strain has excellent proliferation ability under low-temperature fermentation conditions.

[0095] Example 3: Preparation of solid inoculum of Bacillus psychrophilus SL-1 (using cow dung and straw as sources) This embodiment provides a method for preparing a solid bacterial agent loaded with psychrophilic bacillus SL-1 by cross-linking and embedding sodium alginate and calcium chloride using biochar as a carrier.

[0096] 1. Experimental Method: (1) Preparation of biochar carrier: Cow dung and straw were used as raw materials (precursors), dried in an oven at 60℃ to constant weight, pulverized, and passed through a 20-mesh sieve. They were then placed in a muffle furnace and pyrolyzed at 400-450℃ for 5-6 hours under anaerobic conditions. After natural cooling, they were removed, pulverized again, and passed through an 80-mesh sieve to obtain cow dung-derived biochar and straw-derived biochar. The obtained biochars have rich pore structures and specific surface areas ≥3 m². 2 / g.

[0097] (2) Preparation of solid microbial agents: Using the above-prepared cow dung biochar and straw biochar as carriers, biochar was prepared in parallel according to the following steps: 1) Prepare a suspension of Psychrophilic Bacillus SL-1 (viable count 1×10⁻⁶) according to the method in Example 2. 9 (CFU / mL) 2) Mix the biochar from each of the above sources with the bacterial suspension at a mass-to-volume ratio of 1:10 (w / v), and incubate in a shaker at 30℃ and 120r / min for 2 hours to allow the bacteria to fully adsorb the mixture, thus obtaining a mixture of two biochar-bacterial cells. 3) Prepare a 2% (w / v) sterile sodium alginate solution. Mix the above mixture with the sodium alginate solution at a volume ratio of 1:1 and stir for 30 min. 4) Cross-linking and curing were performed using a 4% sterile calcium chloride solution. After standing for 12 hours, the mixture was washed and dried at low temperature.

[0098] Finally, solid microbial inoculants derived from cow dung (NFFHT) and straw (JGFHT) were prepared. Testing showed that both had an effective viable bacteria count ≥5×10⁻⁶. 8 CFU / g.

[0099] 2. Experimental Results and Analysis: Testing revealed that the effective viable count of the prepared psychrophilic bacillus SL-1 solid inoculum was ≥5×10⁻⁶. 8 CFU / g. The solid bacterial agent was observed using scanning electron microscopy (SEM) (see SEM results for reference). Figure 3 The images fully demonstrate the compatibility between the carrier's rich porous structure and the bacteria, showing that a large number of microorganisms successfully attached to and colonized the carrier structure, providing morphological evidence for the high-efficiency survival of solid bacterial agents.

[0100] Example 4: Evaluation of the effect of Bacillus psychrophilus SL-1 and its solid inoculum on tailings improvement in high-altitude areas This embodiment aims to investigate the application effects of the pure liquid bacterial suspension, cow dung-derived solid bacterial agent, and straw-derived solid bacterial agent provided by the present invention in the actual tailings remediation and soil physicochemical property improvement in high-altitude areas.

[0101] 1. Experimental Method: (1) Pot experiment design: The experiment was conducted from late June to late August 2025 to evaluate the effects of different improvement measures on the physicochemical properties and microbial community composition of tailings substrate. Five treatment groups were set up (two control groups and three experimental groups), with three replicates per group. The specific groupings are as follows: Control group (O): pure tailings, with an equal volume (20 mL / kg) of sterile water added; Control group (CK): Pure tailings were inoculated with commercially available phosphorus and potassium solubilizing microbial fertilizer (a mixture of Bacillus and Pseudomonas), at a rate of 20 mL / kg of target substrate (tailings). Experimental group (J): Pure tailings were inoculated with pure psychrophilic bacillus SL-1 bacterial suspension at a rate of 20 mL / kg of target matrix (tailings). Experimental group (NFFHT): Pure tailings with added cow manure-derived solid microbial agent, at an application rate of 10 g / kg of target substrate (tailings). Experimental group (JGFHT): pure tailings with added straw-derived solid microbial agent, at an application rate of 10 g / kg of target substrate (tailings).

[0102] (2) Measurement indicators and methods: After treatment and incubation for 60 days, matrix samples were collected and the following physicochemical properties were measured: Available phosphorus: molybdenum-antimony colorimetric method; Available potassium: Ammonium acetate extraction-flame photometry; Organic carbon: potassium dichromate-sulfuric acid oxidation method; Alkaline nitrogen hydrolysis: alkaline hydrolysis diffusion method.

[0103] Simultaneously, the genome of the samples was extracted for high-throughput sequencing analysis of the microbial community structure.

[0104] 2. Experimental Results and Analysis: (1) Improvement effect on physicochemical indicators: After 60 days of cultivation, the results of the determination of available phosphorus, available potassium, available nitrogen, and organic carbon content in the soil (tailings) of each treatment group are shown in the table below (refer to Table 1): Table 1. Measurement results of each experimental group

[0105] Results analysis: Compared with the control group (O), the tailings treated with pure bacterial solution (J), cow manure-derived solid microbial agent (NFFHT) and straw-derived solid microbial agent (JGFHT) showed a comprehensive improvement in overall fertility.

[0106] The CK group had a limited impact on soil nutrient levels. Data showed that, compared to the original soil control (O group), the CK group did not exhibit significant increases in soil organic carbon, available nitrogen, available phosphorus, and available potassium content after the incubation period; the values ​​of each indicator were essentially at the same level as the O group. This indicates that the microorganisms inoculated in the CK group under high-altitude and low-temperature conditions failed to effectively promote the transformation of soil organic matter and nitrogen mineralization and fixation, and had a weak effect on the activation and release of phosphorus and potassium.

[0107] Experimental group J showed the best performance in increasing the organic carbon content of tailings, significantly enriching the soil carbon pool and improving the physicochemical basis.

[0108] The experimental group NFFHT showed the most significant activation effect on alkaline nitrogen and readily available potassium, effectively improving the direct supply capacity of nitrogen and potassium in tailings.

[0109] The experimental group JGFHT showed the best promoting effect on the release and conversion of available phosphorus in tailings.

[0110] The above results indicate that the three treatment methods can respectively target and optimize the carbon pool structure, nitrogen supply level and phosphorus availability, achieving precise improvement of tailings in high-altitude areas.

[0111] (2) Effect of improving microbial community structure: Therefore, the microbial communities of O (control group), J (pure bacterial suspension), NFFHT (cow dung-derived solid), and JGFHT (straw-derived solid) before and after treatment at the phylum level (reference) Figure 4 ) and genus level (reference) Figure 5 The data shows the changes in ) Pure bacterial agent treatment (Group J): Psychrophilic Bacillus ( Psychrobacter The relative abundance of the species increased significantly and stabilized as the absolutely dominant genus, demonstrating that the exogenous pure strains screened in this invention can successfully colonize, occupy ecological niches, and exhibit strong survival competitiveness in barren, extreme high-altitude tailings environments.

[0112] Solid compound microbial agent treatment (NFFHT and JGFHT group): formed a... Niallia , Pseudarthrobacter The biochar carrier facilitated the introduction of target bacterial strains, resulting in a more complex and diverse microbial community. This indicates that, mediated by the biochar carrier, these two solid bacterial agents not only successfully introduced the target strains but also recruited and formed a specific and rich multifunctional microbial community.

[0113] Conclusion: The data in this embodiment clearly distinguishes the preferred application scenarios for different forms of microbial agents: liquid pure microbial agents (Group J) are more suitable for scenarios that urgently need to increase the total carbon pool of the soil or target functional enhancement; while composite solid microbial agents with biochar as the carrier (NFFHT / JGFHT group) are more suitable for comprehensive matrix improvement scenarios that require multi-species synergistic ecological restoration and comprehensive enhancement of nitrogen, potassium, and phosphorus mineral nutrient release.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of psychrophilic bacillus, characterized in that, The psychrophilic bacillus is a psychrophilic bacillus ( Psychrobacter SL-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34780.

2. A microbial inoculant, characterized in that, It contains the psychrophilic bacillus as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, The microbial agent can be a liquid agent or a solid agent.

4. The microbial agent as described in claim 3, characterized in that, When the microbial agent is a liquid agent, the liquid agent is a bacterial suspension obtained by fermenting and culturing the psychrophilic bacillus at a temperature of 4℃ to 18℃; and / or, When the microbial agent is a solid agent, the solid agent includes a carrier and the psychrophilic bacillus loaded on the carrier.

5. The microbial agent as described in claim 4, characterized in that, The carrier is biochar.

6. The microbial agent as described in claim 5, characterized in that, The solid bacterial agent is prepared by cross-linking and immobilizing a suspension of the psychrophilic bacillus with biochar, followed by encapsulation with sodium alginate and calcium chloride; and / or, The biochar is cow dung-derived biochar and / or straw-derived biochar; and / or, The biochar is produced by pyrolysis of cow dung or straw at 400℃ to 450℃.

7. A method for preparing the microbial inoculant as described in claim 5 or 6, characterized in that, include: Provide a bacterial suspension of the aforementioned psychrophilic bacillus; The bacterial suspension is mixed with the biochar and subjected to adsorption treatment to obtain an adsorption mixture; The adsorption mixture was gelled and immobilized using sodium alginate solution and calcium chloride solution to obtain the solid bacterial agent.

8. The method for preparing the microbial inoculant as described in claim 7, characterized in that, The ratio (v / w) of the bacterial suspension to the biochar is 1:(5-20); and / or, The sodium alginate solution has a mass fraction of 2%; And / or, The calcium chloride solution has a mass fraction of 4%. And / or, The gelation and embedding fixation process includes: uniformly mixing the adsorbed mixture with a 2% (w / v) sterile sodium alginate solution at a volume ratio of 2:1 to 1:1.5 to obtain a mixed colloidal solution; crosslinking the mixed colloidal solution using a 4% (w / v) calcium chloride solution; and / or, The bacterial suspension was obtained by fermentation culture of the psychrophilic bacillus at a temperature of 4°C to 18°C.

9. The application of the psychrophilic bacillus as described in claim 1, or the microbial agent as described in any one of claims 2 to 6, in soil improvement or tailings ecological restoration.

10. The application as described in claim 9, characterized in that, The soil improvement or tailings ecological restoration environment is an area with an altitude of not less than 1500m; and / or, The application includes methods for increasing the content of target elements in soil or tailings; wherein the target elements include at least one of available phosphorus, available potassium, alkaline nitrogen, and organic carbon; and / or, When applying the liquid microbial agent, the application rate is 10 mL / kg to 30 mL / kg of the target substrate; and / or, When applying the solid microbial agent, the application rate is 10 g / kg of the target substrate; the target substrate is soil or tailings.

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