A microbial liquid compound microbial inoculant and biological mineral fertilizer with coal gangue activation and heavy metal treatment

Abstract

This invention belongs to the field of bio-mineral fertilizer technology, specifically relating to a microbial liquid compound agent and bio-mineral fertilizer that combine coal gangue activation and heavy metal remediation. The microbial liquid compound agent comprises the following functional strains prepared through three-stage sequential inoculation and fermentation using electrochemical low-Hertz water: *Pseudomonas putida* (a remedial bacterium), *Azotobacter chrysogenum* (a nitrogen-fixing bacterium), *Azospirillum brasiliensis* (a growth-promoting bacterium), *Pseudomonas fluorescens* (a phosphate-solubilizing bacterium), *Bacillus subtilis* (a biocontrol bacterium), *Bacillus mucilaginosus* (a potassium-solubilizing bacterium), *Bacillus megaterium* (a silica-solubilizing bacterium), and *Trichoderma echinococcus* (a synergistic bacterium). The bio-mineral fertilizer is prepared using the aforementioned microbial liquid compound agent, with coal gangue powder as the main raw material. The scientific combination of different strains provided by this invention achieves efficient improvement of heavy metal-contaminated land and increased crop yields. Simultaneously, it transforms coal gangue into a valuable resource, solving the pollution problem caused by the large-scale accumulation of coal gangue, and possesses significant economic and social value.

Description

Technical Field

[0001] This invention belongs to the field of bio-mineral fertilizer technology, specifically relating to a microbial liquid compound agent and bio-mineral fertilizer that combines coal gangue activation and heavy metal treatment. Background Technology

[0002] Coal gangue, an unavoidable solid waste generated during coal mining and processing, causes a series of environmental pollution problems due to its long-term accumulation. These include land occupation, heavy metal leakage polluting soil and groundwater, soil acidification and desertification caused by high sulfur content, and disruption of regional ecological balance. Furthermore, coal gangue contains a silicate mineral framework and slow-release mineral elements (Si, K, Ca, etc.). Mountains of coal gangue not only occupy vast amounts of land resources, but also, over time, weathering, leaching, and potential spontaneous combustion can all trigger a series of environmental pollution problems, such as air pollution and ecological damage. Therefore, the resource utilization of coal gangue is particularly urgent and important. In recent years, the industry has been actively exploring how to turn coal gangue into a valuable resource. Some reports have suggested using microorganisms to activate the aluminosilicate mineral framework and slow-release mineral elements in coal gangue, thereby enabling the effective utilization of the minerals in coal gangue. However, the utilization efficiency of the existing aluminosilicate mineral framework and slow-release mineral elements in coal gangue is not high.

[0003] Coal gangue contains various heavy metals, including toxic components such as lead (Pb), chromium (Cr), mercury (Hg), arsenic (As), and cadmium (Cd). When washed by rainwater, the soluble components of these heavy metals seep into the soil and groundwater, causing heavy metal pollution. Coal gangue also contains organic sulfur; long-term accumulation and high sulfur content induce soil acidification and desertification, disrupting the regional ecological balance. Furthermore, dust generated from spontaneous combustion or stockpiling of coal gangue carries heavy metals, which enter the soil through dustfall or rainfall. Therefore, areas with coal gangue accumulation pose a high risk of heavy metal pollution, encroaching on land resources. If crops are planted on land with coal gangue accumulation, heavy metals may enter the food chain through plant absorption, threatening human health.

[0004] With the rapid development of modern agriculture, single-function microbial agents for treating heavy metal pollution or activating coal gangue have been reported. However, single-function agents for treating heavy metal pollution usually only target specific heavy metals and have limited ability to remediate soils with multiple pollutions. Single-function coal gangue activating bacteria cannot simultaneously meet the multiple functions required for coal gangue activation (such as nitrogen fixation, phosphorus solubilization, potassium solubilization, etc.), resulting in low activation efficiency. The composition of coal gangue varies greatly in different regions, and single agents may not be able to adapt to all physicochemical conditions (such as pH, heavy metal content, etc.), affecting their stability and effectiveness. In addition, single agents are difficult to colonize in complex soil environments and are easily inhibited by competition from native microorganisms, leading to a rapid decline in viable bacteria count. To address the shortcomings of single-strain microbial agents, some reports have described the use of compound microbial agents for the remediation of coal gangue contaminated areas. These agents achieve functional complementarity, enhanced environmental adaptability and stability, and improved systemic remediation capabilities through the synergistic effect of multiple microbial species. However, the survival, establishment, and interaction with plant roots of compound microbial agents in coal gangue contaminated areas are uncertain, making it difficult to guarantee the desired targeted effects such as growth promotion and disease prevention every time. Furthermore, the soil in coal gangue areas may be contaminated with heavy metals, further inhibiting microbial activity. Additionally, the growth habits (such as requirements for pH, humidity, and nutrients) of different microbial species in compound microbial agents vary significantly; indiscriminate mixing may lead to mutual inhibition between species, reducing overall activity and affecting the remediation effect. Furthermore, the industrial production of compound microbial agents faces severe challenges: if each strain is fermented individually, the process is complex, requiring significant equipment investment and energy consumption; if mixed fermentation is used, the different strains' requirements for culture medium composition and fermentation conditions easily lead to uneven growth rates, competition for nutrients, and the production of inhibitory substances, causing the slow-growing bacteria to be suppressed or even die. This results in an imbalanced microbial community structure, functional degradation, and even off-odors or toxins in the final product, seriously affecting product consistency and safety. Therefore, existing methods for reusing coal gangue have significant limitations. They fail to fully exploit the natural mineral value of coal gangue and soil parent material (its potassium, silicon, phosphorus, and other nutrients are not effectively activated), and compound microbial agents struggle to survive in complex polluted environments, exhibiting suppressed activity and unstable treatment effects. Moreover, industrial preparation is prone to problems such as microbial community imbalance, low yield, and difficulty in controlling product quality. Summary of the Invention

[0005] This invention addresses two major industry pain points: first, the severe environmental pollution caused by the accumulation of large amounts of coal gangue; and second, the difficulty in survival, suppressed activity, and fluctuating treatment effects of existing compound microbial agents in complex environments such as coal gangue pollution, as well as the tendency for their industrial preparation process to suffer from imbalanced microbial community structure, low production efficiency, and inconsistent product quality, making it difficult to achieve precise optimization of various process conditions. This invention provides a microbial liquid compound agent and bio-mineral fertilizer that combines coal gangue activation and heavy metal treatment, thus solving the above problems.

[0006] The technical solution claimed by this invention is as follows:

[0007] A microbial liquid composite inoculant that combines coal gangue activation and heavy metal remediation comprises the following functional strains prepared by three-stage sequential inoculation and fermentation using electrochemical low-hertz water (small molecule cluster water): *Pseudomonas putida* (a restorative bacterium), *Azotobacter chrysogenum* (a nitrogen-fixing bacterium), *Azospirillum brasiliensis* (a growth-promoting bacterium), *Pseudomonas fluorescens* (a phosphate-solubilizing bacterium), *Bacillus subtilis* (a biocontrol bacterium), *Bacillus mucilaginosus* (a potassium-solubilizing bacterium), *Bacillus megaterium* (a silica-solubilizing bacterium), and *Trichoderma echinococcus* (a synergistic bacterium). The inoculation and fermentation preparation includes the following steps:

[0008] S1: Add electrolytic low-hertz water to the sterilized main fermenter, and add the pre-mixed seed liquid of repair bacteria, nitrogen-fixing bacteria and growth-promoting bacteria. Turn on the stirring and adjust the fermentation conditions to the first-order fermentation conditions: pH=6.5-7.0, dissolved oxygen content of 20-30%, temperature of 30℃±0.5℃, humidity of the top space of the fermenter of 90%-92%, fermentation time of 23-25 ​​hours;

[0009] S2: After the S1 stage, adjust to the second-sequence fermentation conditions, and add pre-mixed phosphate-solubilizing bacteria and biocontrol bacteria seed liquid to the main fermenter, and add coal gangue powder to the main fermenter, continue stirring, and adjust the fermentation conditions to the second-sequence fermentation: pH=6.8±0.2, dissolved oxygen 30-40%, temperature 30℃±0.5℃, humidity of the top space of the fermenter 91%-93%, fermentation time 17-19 hours;

[0010] S3: After the fermentation of S2 stage is completed, the pre-mixed seed liquid of potassium-solubilizing bacteria, silica-solubilizing bacteria and synergistic bacteria is added to the main fermenter, and stirring is continued. The fermentation conditions are adjusted to the third time sequence: pH=7.0±0.2, dissolved oxygen content is 40-50%, temperature is 30℃±0.5℃, humidity of the top space of the fermenter is 85%-88%, and fermentation time is 35-37 hours.

[0011] When the total viable bacteria count in the main fermenter is ≥2×10 10 Fermentation ends when CFU / mL is reached.

[0012] Preferably, the volume ratio of the seed liquid of the repair bacteria, nitrogen-fixing bacteria, growth-promoting bacteria, phosphate-solubilizing bacteria, biocontrol bacteria, potassium-solubilizing bacteria, silica-solubilizing bacteria and synergistic bacteria is 2:3:1:1:1:1:1:1.

[0013] Preferably, during the S1 stage, when fermentation reaches the middle of the logarithmic growth phase, nutrient factors citric acid and sodium molybdate, along with a low concentration of Cr in the stress-inducing solution, are added. 6+ After fermentation begins in stage S2 and the pH of the system stabilizes, glycerol and coal gangue powder are added as nutrients. When stage S3 is started, potassium feldspar powder and humic acid are added as nutrients.

[0014] Preferably, the end of stage S1 is marked by: the total bacterial count in the main fermenter ≥ 1 × 10⁻⁶. 9 CFU / mL, heavy metal adsorption rate ≥60%, nitrogenase activity ≥20 nmol C2H4 / (mL·h), IAA content ≥10 mg / L; the end of the S2 phase is marked by: total bacterial count ≥1.5 × 10⁻⁶. 9 The CFU / mL concentration in the supernatant was ≥50 mg / L, and the Bacillus subtilis spore count was ≥90%. The end of the S3 phase was marked by a total viable count ≥2 × 10⁻⁶. 10 The total number of viable bacteria is CFU / mL, with a potassium feldspar decomposition rate ≥35%, a silicon leaching rate ≥25%, and a coal gangue organic matter conversion rate ≥30%; the total number of viable bacteria includes fungal spores and bacteria.

[0015] Preferably, the preparation and mixing process of the seed liquid in S1-S3 includes the following steps:

[0016] Step 1: Pretreatment: Each strain was inoculated into a special culture medium and cultured at 30℃ for 24-48 hours until the logarithmic phase; the repair bacteria were cultured in LB medium with 0.01% cadmium ion medium; the nitrogen-fixing bacteria were cultured in Assambe nitrogen-free medium; the phosphate-solubilizing bacteria were cultured in KB medium; the biocontrol bacteria were cultured in LB medium; the potassium-solubilizing bacteria were cultured in potassium-solubilizing medium; the silica-solubilizing bacteria were cultured in beef extract peptone medium with 2% silica mineral powder medium; the Trichoderma echinosporum was induced to culture in PDA medium containing 0.5% sand; the special culture medium was prepared with electrolytic low-hertz water and sterilized at 121℃ for 20 minutes.

[0017] Step 2: Primary Seed Tank Cultivation: Configure independent primary seed tanks and add the strains cultivated in Step 1 to different independent primary fermenters. After cultivation for a certain period, obtain seed solutions for single strains. The cultivation conditions for repair bacteria and nitrogen-fixing bacteria are: temperature 30℃±0.5℃, humidity at the top of the fermenter 88-90%, dissolved oxygen 20-30%, and cultivation time 24 hours. The cultivation conditions for phosphate-solubilizing bacteria, potassium-solubilizing bacteria, and silica-solubilizing bacteria are: temperature 30℃±0.5℃, humidity at the top of the fermenter 90-92%, and dissolved oxygen 30-40%. The cultivation conditions for growth-promoting bacteria and biocontrol bacteria are: temperature 30℃±0.5℃, humidity at the top of the fermenter 85-88%, and dissolved oxygen 25-35%. The cultivation conditions for synergistic bacteria are: temperature 30℃±0.5℃, pH 5.5-6.0, humidity at the top of the fermenter 70-5%, and dissolved oxygen 40-50%.

[0018] Step 3: Secondary premix tank culture: Take the seed liquid of the single strain obtained in Step 2, mix it with the seed liquid to be mixed in a certain proportion, keep it at 30℃ for 30 minutes to obtain the seed liquid of the mixed strain.

[0019] Preferably, S1 contains citric acid, sodium molybdate, and a low concentration of Cr.6+ The treatment employs pulse dosing, with each addition being 1 / 3 of the total required amount, spaced 2 hours apart, for a total of 3 times. The pulse dosing is performed using a slow-flow injection method, completed within 10 minutes. One hour after each injection of citric acid and sodium molybdate, a low concentration of Cr is slowly injected. 6+ One-third of the total liquid demand must be completed within 5 minutes.

[0020] In S2, glycerol and coal gangue powder are added simultaneously. The glycerol is continuously injected using a peristaltic pump, and the coal gangue powder is added in batches through a sterile crushing and feeding device.

[0021] In S3, potassium feldspar powder and humic acid are added simultaneously. The potassium feldspar powder is added in batches through a sterile feeding device, while the humic acid is continuously injected through a peristaltic pump.

[0022] The present invention also provides a bio-mineral fertilizer that combines coal gangue activation and heavy metal remediation, which is prepared using the aforementioned microbial liquid compound agent that combines coal gangue activation and heavy metal remediation.

[0023] Preferably, the method for preparing the bio-mineral fertilizer includes:

[0024] A1: The aforementioned microbial liquid compound inoculant, 80-mesh coal gangue powder, wheat bran, soybean meal, and electrolytic low-hertz water are added to the mixing system. After the mixture is evenly stirred, it is conveyed to the solid-state fermentation system production line through an intelligent material distribution system for the first solid-state fermentation. The solid-state fermentation system automatically adjusts the temperature during the fermentation process through a temperature sensor linked to the intermittent micro-burst oxygen frequency: the initial stage temperature is 42-45℃, maintained for 22-24 hours; the peak stage temperature is 60-65℃, maintained for 46-48 hours; and the final stage temperature is 40-45℃, maintained for 22-24 hours.

[0025] A2: The material obtained from the first solid-state fermentation in A1 is rearranged into the aging and fermentation production line for a second aging and fermentation through an intelligent material distribution system: the naturally piled material is deeply decomposed by utilizing the residual heat and residual microorganisms of the material, and the fermentation ends after 10-12 days.

[0026] Preferably, the completion of the first solid-state fermentation in A1 is marked by: the temperature maintaining at the final stage temperature for 24 consecutive hours, material moisture content ≤40%, material color dark brown, no odor, loose to the touch, and no stickiness; the completion of the second aging fermentation in A2 is marked by: the material naturally drying to a moisture content of 15-29%, uniform dark brown color, loose texture, earthy smell without other odors, pH 6.5-7.5, and beneficial live bacteria count ≥200 million / g; A1 consists of solid components: coal gangue powder, wheat bran, soybean... The total amount of meal is 100%, and the proportions of each solid component are as follows: 90-95% 80-mesh coal gangue powder, 3-8% wheat bran, and 4-10% soybean meal. Among them, the initial moisture content of coal gangue powder is 5-9%, and the initial moisture content of wheat bran and soybean meal is 8-12%. After the above solid components are mixed evenly, electrochemical low-hertz water is added and stirred thoroughly. At the same time, a microbial liquid compound inoculant is added simultaneously during the stirring process, with the addition amount calculated as 0.8-1.2% of the total solid substrate, so that the total moisture content of the material reaches 50-60%.

[0027] Beneficial effects

[0028] This invention provides a microbial liquid compound inoculant and bio-mineral fertilizer that combines coal gangue activation and heavy metal remediation. Through a grouped and time-sequential fermentation design, eight strains with different fermentation conditions and mutual growth within the same time sequence and metabolic complementarity between different time sequences are organically combined. By designing the optimal fermentation conditions for each time sequence, this invention not only solves the problems of microbial imbalance, low production efficiency, inconsistent product quality, and difficulty in precisely optimizing various process conditions that are prone to occur in the industrial preparation of compound inoculants, but also ensures that the prepared inoculant can survive well in the complex environment of coal gangue pollution, effectively exert its activity, and achieve stable remediation results, thereby effectively solving the severe environmental pollution problem caused by coal gangue accumulation.

[0029] Specifically, in the first sequence of the liquid compound microbial agent of this invention, repair bacteria, nitrogen-fixing bacteria, and growth-promoting bacteria are selected. The nitrogen-fixing bacteria and growth-promoting bacteria preferentially convert atmospheric N2 into ammonia nitrogen through nitrogen fixation, increasing the nitrogen content of the fermentation system to 1.2-1.5 g / L within 22-24 hours. This provides a basic nitrogen source for protein synthesis by subsequent strains. The simultaneously inoculated repair bacteria grow rapidly on the basis of this nitrogen source. After application of this compound microbial agent, the repair bacteria can reduce / chelate Cd. 2+This method effectively removes heavy metals and reduces bioavailability, making it suitable for scenarios involving heavy metal pollution associated with coal gangue. Furthermore, the addition of remediation bacteria in the first phase reduces the heavy metal toxicity from coal gangue after the subsequent addition of the compound microbial agent to the soil. In addition, in the first phase, nitrogen-fixing bacteria secrete organic acids such as succinic acid and citric acid, precisely adjusting the initial pH of the system from 7.5-8.0 to 6.8±0.2. This pH value directly meets the growth requirements of the inoculated strains in the second phase, eliminating the need for additional pH adjustments. In the second phase, phosphate-solubilizing bacteria and biocontrol bacteria are selected. Supported by the nitrogen source accumulated in the early stages, the phosphate-solubilizing bacteria rapidly multiply. After the compound microbial agent is applied to the soil, the organic acids secreted by the phosphate-solubilizing bacteria can efficiently dissolve insoluble phosphorus such as Ca3(PO4)2 in coal gangue. The phosphate-solubilizing bacteria can also further assist in fine-tuning the pH and optimizing the growth environment. The biocontrol bacteria also rapidly multiply using nitrogen sources, secreting antimicrobial peptides such as isochorin to inhibit soil-borne pathogens (such as Fusarium, with an inhibition rate ≥80%), synergistically enhancing the stability of the entire microbial community system. The third inoculation sequence involves potassium-solubilizing bacteria, silicate-solubilizing bacteria, and synergistic bacteria: Potassium-solubilizing and silicate-solubilizing bacteria rely on the nitrogen and phosphorus nutrients accumulated in the first two stages to synthesize decomposing enzyme systems. After the inoculant is applied, potassium-solubilizing bacteria secrete organic acids / phytases to dissolve insoluble phosphorus and produce silicate enzymes to decompose aluminosilicates in coal gangue, simultaneously releasing potassium and silicon. Silicate-solubilizing bacteria specifically secrete silicate enzymes to degrade coal gangue, releasing available silicon. As a synergistic bacterium, *Trichoderma echinosporum* secretes cell wall hydrolases such as chitinases to decompose organic matter and secretes oxalic acid to enhance heavy metal chelation and mineral dissolution. During this stage, mechanical stirring is required, while dissolved oxygen is maintained at 40-50% to provide conditions for *Trichoderma echinosporum* to efficiently produce oxalic acid and citric acid, ensuring that these acidic substances can fully contact the minerals and heavy metals in the soil after application. After experimental verification following the application of the compound microbial agent, the compound microbial agent prepared by the above method showed excellent performance upon application: high survival rate of the microbial community, with each strain fully exerting its targeted activity, effectively solving the problems of existing compound microbial agents having difficulty surviving in complex polluted environments, suppressed activity, and unstable treatment effects. Simultaneously, the liquid microbial compound agent, combined with coal gangue to produce 80-mesh coal gangue powder (90-95% coal gangue powder), forms a bio-mineral fertilizer, effectively activating the effective components in the coal gangue, turning coal gangue from waste into treasure, and solving the problem of severe environmental pollution caused by excessive coal gangue accumulation. The three stages must be strictly followed in sequence, with the following core advantages: First, it avoids the inhibition of slow-growing bacteria by rapidly growing bacteria: Bacteria inoculated in the first and second stages can quickly occupy ecological niches and do not produce strong inhibitory metabolites; Trichoderma echinococci inoculated in the third stage (generation time 6-8 hours) grows more slowly, at which point the system has already established a stable nutrient distribution pattern (e.g., nitrogen source mainly supplies bacteria, and 30% carbon source is reserved for fungi), which can avoid the inhibition of fungi by excessive bacterial proliferation. If inoculated simultaneously, bacteria will occupy more than 90% of nutrient sites within 12 hours, resulting in a Trichoderma echinococci spore germination rate of <15%.Secondly, cross-antagonistic effects of metabolites are avoided: While chitinase secreted by *Trichoderma echinosporum* may damage bacterial cell walls, by the third inoculation time, the bacteria in the earlier stages have already entered a stable phase (cell wall structure integrity increased by 40%), and the antimicrobial peptides produced by potassium-solubilizing bacteria can form an "antagonistic balance" with the chitinase of *Trichoderma*—the antimicrobial peptides inhibit excessive enzyme production by *Trichoderma*, while the chitinase inhibits the growth of other microorganisms. If the stage order is reversed (e.g., *Trichoderma* is inoculated first), the secreted chitinase will cause a 60% decrease in the viable count of the earlier bacteria within 24 hours, completely losing nitrogen fixation and phosphorus solubilization functions. In summary, through precise timing control, competitive inhibition between strains can be effectively avoided, achieving orderly propagation of different functional bacterial communities. This successfully solves the problems of bacterial imbalance, low yield, and difficulty in controlling product quality that easily occur in the industrial preparation of compound microbial agents, achieving optimized adaptation of various process conditions.

[0030] This invention achieves three core objectives simultaneously through the scientific formulation and targeted domestication of various functional microorganisms: transforming coal gangue, a mining waste, into high-quality mineral fertilizer raw materials, thus realizing the high-value utilization of resources; efficiently remediating soil heavy metal pollution and reducing the bioavailability of heavy metals; and improving the physical and chemical properties and micro-ecological structure of the soil to create a healthy soil environment for crop growth, ultimately achieving the integrated benefits of "pollution control - turning waste into treasure - increasing agricultural production".

[0031] This invention focuses on a dual core of "environmental pollution control + high-value utilization of waste," rather than simply addressing pollution remediation. It provides a microbial liquid compound agent and bio-mineral fertilizer that combines the functions of coal gangue activation, heavy metal remediation, and soil mineral replenishment. Through the scientific formulation of various functional microorganisms, it effectively controls environmental pollution caused by coal gangue stockpiles, reduces the bioavailability of heavy metals, and improves soil acidification. Simultaneously, it deeply activates the natural mineral nutrients in coal gangue, transforming this mining waste into high-quality bio-mineral fertilizer raw materials. This replenishes scarce soil minerals for agricultural production, contributing to healthy and sustainable soil development. Ultimately, it achieves the integrated goal of "pollution control, waste-to-treasure transformation, and agricultural efficiency enhancement," providing an economically feasible solution for the resource utilization of mining waste and farmland soil improvement.

[0032] In this invention, the microbial liquid compound inoculant employs a metabolic complementary design, wherein: oxalic acid secreted by *Trichoderma echinosporum* provides a carbon source for *Azotocinobacter chrysogenum*, and *Azotocinobacter chrysogenum* releases NH4. + It promotes the growth of fungal strains; Bacillus subtilis produces surfactants that enhance biofilm permeability, increasing the enzyme production efficiency of Trichoderma echinosporum by 35%; the entire fermentation process can meet the growth requirements of various strains without the need for additional culture medium and other nitrogen sources.

[0033] This invention utilizes a single, independent main fermenter for different fermentation sequences, avoiding idle tanks and reducing the probability of cross-contamination and production costs. Furthermore, all three stages employ electrochemical low-Hertz water for fermentation. This electrochemical low-Hertz water has a permeability more than 30% higher than ordinary water, strong dissolving power, and can quickly pass through cell membrane water channels (only 0.5 nanometers in diameter), carrying nutrients into cells and accelerating the excretion of metabolic waste. Simultaneously, the electrochemical low-Hertz water also enhances the permeability of microbial cell membranes, promotes the transmission of intercellular signaling molecules (such as quorum sensing substances), and optimizes the metabolic network of the complex microbial community. The small-cluster water is weakly alkaline (pH 7.5-8.0), which can neutralize acidic metabolites (such as lactic acid and acetic acid) produced during fermentation, maintaining the optimal pH environment for enzymatic reactions. Furthermore, electrochemically treated low-Hertz water promotes nutrient exchange among different microbial strains, improves overall fermentation efficiency, and achieves nutrient sharing and metabolic complementarity. It can neutralize acidic metabolites, optimize the fermentation environment, maintain the optimal growth pH for the complex microbial community, and reduce metabolic inhibition. It also enhances stress resistance, strengthens cell membrane stability, and improves the survival rate of the complex microbial agent under high temperature and high salt stress conditions. Therefore, electrochemically treated low-Hertz water possesses unique physicochemical advantages in microbial fermentation, significantly improving microbial metabolic efficiency and product synthesis capabilities.

[0034] During the S1 stage, when fermentation reaches the middle of the logarithmic growth phase, nutrient factors citric acid, sodium molybdate, and a low concentration of Cr are added. 6+ The citric acid chelates heavy metals in coal gangue, enhancing the adsorption capacity of the remediation bacteria. Simultaneously, it adjusts the pH of the system to 6.5-7.0, thus adapting it to the growth of both remediation and nitrogen-fixing bacteria. Sodium molybdate, as a core cofactor of nitrogenase, maintains nitrogenase homeostasis; its concentration is consistent with the basal culture medium to avoid metal toxicity. The low-concentration Cr... 6+ The induced repair bacteria (Pseudomonas putidae) activate the chrA stress-resistance gene, promote the secretion of heavy metal adsorption proteins (such as metallothionein), and increase the tolerance threshold of the bacteria to complex heavy metals in coal gangue. After the fermentation of stage S2 begins and the pH of the system stabilizes, glycerol and coal gangue powder are added. The glycerol serves as a slow-release carbon source to induce phosphate-solubilizing bacteria and silicate-solubilizing bacteria to produce enzymes. The coal gangue powder provides a phosphorus source substrate for the phosphate-solubilizing bacteria (coal gangue contains insoluble phosphorus) and also helps them adapt to the subsequent organic fertilizer carrier in advance. When stage S3 starts, potassium feldspar powder and humic acid are added. The potassium feldspar powder provides a potassium source for the potassium-solubilizing bacteria, and the humic acid provides a carbon source for Trichoderma echinosporum, while also improving the water and fertilizer retention capacity of the organic fertilizer.

[0035] In S1, citric acid and sodium molybdate are simultaneously added in a pulse (slow-flow injection, completed within 10 minutes), with each addition being 1 / 3 of the total required amount, spaced 2 hours apart, for a total of 3 times. This is to prevent excessively high instantaneous concentrations from inhibiting bacterial growth (e.g., citric acid > 0.03% will cause a 20% decrease in the metabolic rate of nitrogen-fixing bacteria); one hour after each injection of citric acid and sodium molybdate, a low concentration of Cr is slowly injected.6+ One-third of the total liquid requirement (2-5 mg / L) is completed within 5 minutes; in S2, glycerol is continuously injected via a peristaltic pump, and coal gangue powder is added in batches via a sterile pulverizing and feeding device, with both added simultaneously (glycerol provides energy, coal gangue powder provides substrate, and phosphorus activation efficiency is increased by 25%); in S3, potassium feldspar powder is added in batches via a sterile feeding device, and humic acid is continuously injected via a peristaltic pump, with both added simultaneously (humic acid can improve the dispersibility of potassium feldspar powder and increase the contact area by 25%).

[0036] The three groupings and corresponding time sequences designed in the microbial liquid compound inoculant production process provided by this invention are scientifically efficient, ensuring no interspecies antagonism during the fermentation of each strain, that their own propagation is not inhibited, and that the fermentation products of the strains propagating earlier do not adversely affect subsequent strains, but can be efficiently utilized by the strains propagating later to promote growth. Furthermore, due to the rational design, the secondary propagation process of the microbial liquid compound inoculant (i.e., the time-sequential propagation in the main fermenter) only requires the addition of trace amounts of growth factors and electrochemical low-hertz water (small molecule cluster water), eliminating the need for additional carbon and nitrogen sources, thus simplifying the production process and effectively reducing production costs.

[0037] This invention's eight-strain microbial liquid compound inoculant, relying on an innovative process of group formulation and phased sequential fermentation, successfully overcomes the technical bottlenecks in the industrial preparation of compound inoculants, such as imbalanced microbial community structure, low production efficiency, product quality fluctuations, and difficulty in precisely optimizing process conditions. Furthermore, after the inoculant and bio-mineral fertilizer are applied to the soil, the targeted effects of each functional strain can be fully realized. It effectively addresses the pain points of existing compound inoculants in coal gangue-polluted areas, such as difficulty in microbial community survival, suppressed activity, and unstable application effects. Moreover, because bio-mineral fertilizer has universal fertility supplementation and microecological improvement value, it can be widely adapted to various types of arable land, saline-alkali land, and barren land, not limited to coal gangue-polluted areas. In addition, the single-strain targeted function and synergistic effect of the eight strains not only consolidate the treatment and improvement results but also simultaneously achieve multiple values ​​such as coal gangue mineral activation, soil heavy metal detoxicity reduction, and enhanced soil nutrient supply, providing an integrated solution for the resource utilization of mining waste and the improvement of soil quality across the entire region.

[0038] Firstly, *Pseudomonas fluorescens*, *Azospirillum brasiliensis*, and *Bacillus subtilis* can form a core protective network, while *Pseudomonas putida*, as a pioneer species, has strong resistance to heavy metals (it can tolerate Cd). 2+ Pb 2+ Cr 6+(e.g.,) can colonize first, chelating heavy metals through the secretion of siderophores, reducing environmental toxicity, and creating a "safe haven" for subsequent microbial communities; *Azospirillum brasiliensis* proliferates rapidly under the stimulation of root exudates (such as sugars and amino acids), and its nitrogen-fixing activity is highest at pH 8.0, which is perfectly adapted to the alkaline environment of coal gangue. It can produce plant hormones (IAA), promoting the development of pioneer plant roots, while root exudates in turn nourish the microbial community, forming a positive cycle of "microbe-plant"; the biocontrol bacterium *Bacillus subtilis* forms biofilms and secretes lipopeptide antibiotics (such as surfactants), which can inhibit the invasion of pathogenic fungi and encapsulate and protect its own spores and other functional bacteria, resisting drought, ultraviolet radiation and heavy metal toxicity. It can also produce extracellular polysaccharides (EPS), improving the particle structure of coal gangue and enhancing its water and fertilizer retention capacity. It can degrade polycyclic aromatic hydrocarbons (PAHs) in coal gangue, releasing carbon sources for other bacteria to utilize. The process of *Pseudomonas putida* degrading organic pollutants and releasing carbon and nitrogen sources, followed by *Azotrophus brasiliensis* fixing atmospheric nitrogen, and *Bacillus subtilis* providing physical protection and pathogen inhibition, together constitutes a self-sufficient basic micro-ecosystem.

[0039] Secondly, each species possesses a unique stress adaptation strategy, achieving its own independent function through niche differentiation, as shown in Table 1.

[0040] Table 1. Environmental application strategies and key roles of 8 strains

[0041]

[0042] Third, the metabolic cross-feeding mechanism: Carbon source complementarity: The intermediate products such as catechol produced by *Pseudomonas putida* from the degradation of polycyclic aromatic hydrocarbons (PAHs) can be efficiently utilized by *Bacillus subtilis*; while the extracellular polysaccharides secreted by *Bacillus subtilis* can serve as a carbon source for *Trichoderma echinosporum*, forming a carbon source recycling chain. Nitrogen source sharing: The nitrogen fixed by *Azotobacter chamaescoparia* and *Azospirillum brasiliensis* is released into the environment as ammonium salts, allowing heterotrophic bacteria such as *Bacillus megaterium* to directly absorb it, avoiding limitations imposed by nitrogen scarcity on all species and ensuring synergistic function. Signal molecule regulation: The xylanase and cellulase produced by *Trichoderma* can decompose humic precursors in coal gangue, releasing signal molecules such as N-acylhomoserine lactones, thereby activating the quorum sensing (QS) system of other bacteria, significantly enhancing the biofilm formation ability and stress resistance gene expression levels of the bacterial community.

[0043] Fourth, spore protection mechanism: Bacillus subtilis, Bacillus mucilaginosus, and Bacillus megaterium can form spores in the harsh environment of coal gangue (such as high salt and low nutrients), which are like "dormant seeds" that re-germinate when the rainy season arrives or the temperature reaches a suitable range, effectively ensuring the stability of the long-term application effect of the microbial agent.

[0044] Fifth, targeted strategies to address specific stresses caused by coal gangue: Heavy metal stress: Siderophores secreted by *Pseudomonas putida* and *Pseudomonas fluorescens* can efficiently chelate heavy metal ions; *Bacillus subtilis* forms biofilms that can adsorb and fix free heavy metals; *Trichoderma* mycelium reduces the heavy metal content in the environment through enrichment, and reduces the bioavailable concentration of heavy metals through multi-level synergy. High sulfur environment: *Bacillus megaterium*'s silicate-releasing activity can release silicates, which combine with sulfur ions in the environment to form a stable mineral phase, effectively reducing the risk of soil acidification. Organic matter deficiency: Nitrogen-fixing bacteria directly utilize atmospheric nitrogen, without relying on the limited nitrogen source in the soil; phosphorus- and potassium-solubilizing bacteria release phosphorus and potassium nutrients from coal gangue minerals, achieving "mineral-based bacterial growth"; *Trichoderma echinosporum* decomposes residual lignin and cellulose in coal gangue, providing a slow-release carbon source and solving the problem of nutrient deficiency. Temperature fluctuations: The spore structure of Bacillus and the spores of Trichoderma can withstand drastic temperature changes from -20℃ to 50℃, ensuring that the inoculant can maintain its activity under different climatic conditions throughout the year.

[0045] Sixth, the stability of the compound microbial agent is also reflected in the fact that even if some microbial species are inactivated under extreme conditions, other microbial species can still maintain basic ecological functions. After the environment improves, the dormant spores and buds can restore the integrity of the community, forming an elastic body with "redundant design and dynamic balance". Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be further described clearly and completely below in conjunction with specific embodiments of the present invention.

[0047] Unless otherwise specified, the components described in the embodiments of this invention are all readily available to those skilled in the art.

[0048] The first set of embodiments: a microbial liquid compound agent that combines coal gangue activation and heavy metal treatment.

[0049] This set of embodiments provides a microbial liquid composite agent that combines coal gangue activation and heavy metal remediation. It comprises the following functional strains prepared by electrochemical low-Hertz water through a three-stage sequential inoculation and fermentation process: *Pseudomonas putida* (a restorative bacterium), *Azotobacter chrysogenum* (a nitrogen-fixing bacterium), *Azospirillum brasiliensis* (a growth-promoting bacterium), *Pseudomonas fluorescens* (a phosphate-solubilizing bacterium), *Bacillus subtilis* (a biocontrol bacterium), *Bacillus mucilaginosus* (a potassium-solubilizing bacterium), *Bacillus megaterium* (a silica-solubilizing bacterium), and *Trichoderma echinococcus* (a synergistic bacterium). The inoculation and fermentation preparation includes the following steps:

[0050] S1: Add electrolytic low-hertz water (small molecule cluster water) to the sterilized main fermenter, and add 9.8%-10.2% (by fermentation volume) of pre-mixed seed culture of repair bacteria, nitrogen-fixing bacteria, and growth-promoting bacteria. Start stirring and adjust the fermentation conditions to the first-order fermentation conditions: pH=6.5-7.0, dissolved oxygen content of 20-30%, temperature of 30℃±0.5℃, humidity of the top space of the fermenter of 90%-92%, and fermentation for 23-25 ​​hours. In a specific embodiment of the present invention, the seed culture of repair bacteria, nitrogen-fixing bacteria, and growth-promoting bacteria is inoculated at a volume ratio of 2:3:1, and nutrient factors are added. All three require a low-toxicity, medium-nitrogen environment: the remediation bacteria preferentially reduce toxicity, nitrogen-fixing bacteria provide the nitrogen source, and growth-promoting bacteria utilize the nitrogen source to synthesize IAA. This ratio balances toxicity reduction efficiency (remediation bacteria) and nutrient supply (nitrogen-fixing bacteria), reducing interspecies competition. The added nutrients and parameters at this stage are as follows: Citric acid (0.02%): chelates heavy metals in coal gangue, enhancing the adsorption capacity of the remediation bacteria, and simultaneously adjusting the system pH to 6.5-7.0 (suitable for the growth of both remediation and nitrogen-fixing bacteria); Sodium molybdate (10μM): a core cofactor for nitrogenase, maintaining nitrogenase homeostasis; its concentration is consistent with the basal culture medium to avoid metal toxicity; low concentration of Cr... 6+ (2-5 mg / L): The inducing repair bacteria (Pseudomonas putida) activate the chrA stress resistance gene, promote the secretion of heavy metal adsorption proteins (such as metallothionein), and increase the tolerance threshold of the bacteria to complex heavy metals in coal gangue.

[0051] Timing of addition: During the middle of the logarithmic growth phase of fermentation (12-16 hours, OD...) 600 =1.0-1.2), during which the bacterial metabolic activity peaks, cell membrane permeability is high, and the absorption efficiency of nutrient factors and stress inducers is optimal. This can maximize the activation of the heavy metal adsorption genes of the repair bacteria, while avoiding bacterial stress caused by the addition during the lag period.

[0052] Addition method: Citric acid and sodium molybdate are added simultaneously using a pulsed injection (slow-flow injection, completed within 10 minutes), with each addition being 1 / 3 of the total required amount, spaced 2 hours apart, for a total of 3 times. This is to prevent excessively high instantaneous concentrations from inhibiting bacterial growth (e.g., citric acid > 0.03% will cause a 20% decrease in the metabolic rate of nitrogen-fixing bacteria); 1 hour after each injection of citric acid and sodium molybdate, a low concentration of Cr is slowly injected. 6+ One-third of the total liquid requirement (2-5 mg / L) is completed within 5 minutes.

[0053] The end of this stage is marked by: a total viable count of ≥1×10⁻⁶ bacteria in the main fermenter using the plate count method. 9 CFU / mL (fungal spores plus bacteria), nitrogenase activity ≥20 nmol C2H4 / (mL·h) using acetylene reduction method, and Cr content of bacteria repaired using diphenylcarbazide colorimetric method. 6+The reduction rate is ≥20%, and the IAA content is ≥10mg / L (high performance liquid chromatography).

[0054] S2: After the S1 stage, adjust to the second-sequence fermentation conditions. Add 4.8%-5.2% (by fermentation volume) of pre-mixed phosphate-solubilizing bacteria and biocontrol bacteria seed solution to the main fermenter, and add coal gangue powder to the main fermenter. Continue stirring and adjust the fermentation conditions to the second-sequence fermentation: pH=6.8±0.2, dissolved oxygen 30-40%, temperature 30℃±0.5℃, humidity of the top space of the fermenter 91%-93%, fermentation time 17-19 hours. In a specific embodiment of the invention, phosphate-solubilizing bacteria and biocontrol bacteria are inoculated at a 1:1 volume ratio, and nutrient factors are added. At this stage, the phosphate-solubilizing bacteria need to utilize the nutrients from the first stage. Nitrogen sources secrete phosphorus-degrading enzymes, and biocontrol bacteria require phosphorus to synthesize antimicrobial peptides. This ratio can synergistically enhance phosphorus activation efficiency (available phosphorus ≥ 50 mg / L) and biocontrol function (antimicrobial peptides ≥ 4 mg / L). The nutrient factors and parameters added at this stage are as follows: 1. Glycerol (5 mM): continuously added (flow rate 0.5 g / L·h) as a slow-release carbon source to induce phosphate-solubilizing bacteria to synthesize phytase, avoiding local concentrations > 10 mM that inhibit biocontrol bacteria; 2. Coal gangue powder (200 mesh, 5 g / L): added in 3 batches (1 / 3 each time, 4 hours apart) to provide phosphorus source substrate for phosphate-solubilizing bacteria (coal gangue contains insoluble phosphorus) and to allow them to adapt to the subsequent organic fertilizer carrier in advance.

[0055] Timing of addition: After the first stage of fermentation is completed and the system pH is stable (6.8±0.2) (24-36 hours), to avoid pH fluctuations affecting the enzyme production of phosphate-solubilizing bacteria;

[0056] Addition method: Glycerin is continuously injected through a peristaltic pump, and coal gangue powder is added in batches through a sterile crushing and feeding device. Both are added simultaneously (glycerin provides energy, coal gangue powder provides substrate, and phosphorus activation efficiency is increased by 25%).

[0057] The end of this stage is marked by a total bacterial count ≥ 1.5 × 10⁻⁶. 9 CFU / mL (including residual bacteria from the first stage, plate count method), and the effective phosphorus concentration of the supernatant is ≥50mg / L (molybdenum blue colorimetric method), and the spore rate of Bacillus subtilis is ≥90% (spore staining method).

[0058] S3: After the S2 stage fermentation is completed, a pre-mixed seed culture of potassium-solubilizing bacteria, silica-solubilizing bacteria, and synergistic bacteria (7.8%-8.2% fermentation volume) is added to the main fermenter. Stirring continues, and the fermentation conditions are adjusted to the third-order fermentation conditions: pH = 7.0 ± 0.2, dissolved oxygen 40-50%, temperature 30℃ ± 0.5℃, humidity at the top of the fermenter 85%-88%, and fermentation time 35-37 hours. In a specific embodiment of the invention, the seed culture of potassium-solubilizing bacteria, silica-solubilizing bacteria, and synergistic bacteria is inoculated at a volume ratio of 1:1:1, and nutrient factors are added. The potassium-solubilizing / silica-solubilizing bacteria require the nutrients from the first two stages. Nitrogen and phosphorus nutrient synthesis and decomposition enzymes, along with oxalic acid secreted by synergistic bacteria, enhance the decomposition of coal gangue. This ratio can reduce the damage of bacterial cell walls by Trichoderma chitinase (chitinase activity decreases by 40%). The nutrient factors and parameters added at this stage are as follows: 1. Potassium feldspar powder (200 mesh, 10 g / L): added in two portions (6 hours apart) to prevent excessive addition from causing the system viscosity to be >50 cP (dissolved oxygen efficiency decreases by 30%), providing a potassium source for potassium-solubilizing bacteria; 2. Humic acid (0.5%): continuously added (flow rate 0.1 g / L·h), providing a carbon source for Trichoderma echinosporum and simultaneously improving the water and fertilizer retention capacity of organic fertilizer;

[0059] Timing of addition: When the third stage is started (i.e., 0 hours after inoculation with potassium-solubilizing / silicicizing bacteria), coal gangue nutrient substrate is provided to the bacterial community simultaneously;

[0060] Addition method: Potassium feldspar powder is added in batches through a sterile feeding device, and humic acid is continuously injected through a peristaltic pump. Both are added simultaneously (humic acid can improve the dispersibility of potassium feldspar powder and increase the contact area by 25%).

[0061] The end of this stage is marked by a total viable count ≥ 2 × 10⁻⁶. 10 CFU / mL (containing fungal spores; bacteria were counted using LB plates, and Trichoderma were counted using PDA plates), and the potassium feldspar decomposition rate was ≥35% (atomic absorption spectrometry), the silicon dissolution rate was ≥25% (molybdenum blue colorimetric method), and the coal gangue organic matter conversion rate was ≥30% (potassium dichromate method).

[0062] When the total viable count (bacterial count + fungal spore count) in the main fermenter is ≥2×10 10 Fermentation ends when CFU / mL is reached.

[0063] In a specific embodiment of the present invention, the amount of electrochemical low-hertz water (small molecule cluster water) introduced is 76.4%-77.6% of the fermentation volume, and the electrochemical low-hertz water (small molecule cluster water) has a molecular cluster of ≤6 molecules and a pH of 7.0-7.5.

[0064] In a specific embodiment of the present invention, the volume ratio of the seed liquid of the repair bacteria, nitrogen-fixing bacteria, growth-promoting bacteria, phosphate-solubilizing bacteria, biocontrol bacteria, potassium-solubilizing bacteria, silica-solubilizing bacteria and synergistic bacteria is 2:3:1:1:1:1:1:1.

[0065] In a specific embodiment of the present invention, the preparation and mixing process of the seed liquid includes the following steps:

[0066] Step 1: Pretreatment: Each strain was inoculated into its specific culture medium and incubated at 30°C for 24-48 hours until the logarithmic phase. The repair bacteria were cultured in LB medium with 0.01% cadmium ion medium; the nitrogen-fixing bacteria were cultured in Assambe nitrogen-free medium; the phosphate-solubilizing bacteria were cultured in KB medium; the biocontrol bacteria were cultured in LB medium; the potassium-solubilizing bacteria were cultured in potassium-solubilizing medium; the silica-solubilizing bacteria were cultured in beef extract peptone medium with 2% silica mineral powder medium; the Trichoderma echinosporum was induced to culture in PDA medium containing 0.5% sand; the specific culture medium was prepared with electrolytic low-hertz water and sterilized at 121°C for 20 minutes.

[0067] Step 2: Primary Seed Tank Cultivation: Configure independent primary seed tanks and add the strains cultivated in Step 1 to different independent primary fermenters. After cultivation for a certain period, obtain seed solutions of single strains. Specifically: the cultivation conditions for repair bacteria and nitrogen-fixing bacteria are: temperature 30℃±0.5℃, humidity at the top of the fermenter 88-90%, dissolved oxygen 20-30%, and cultivation time 24h; the cultivation conditions for phosphate-solubilizing bacteria, potassium-solubilizing bacteria, and silica-solubilizing bacteria are: temperature 30℃±0.5℃, humidity at the top of the fermenter 90-92%, dissolved oxygen 30-40%, and cultivation time 20h; the cultivation conditions for growth-promoting bacteria and biocontrol bacteria are: temperature 30℃±0.5℃, humidity at the top of the fermenter 85-88%, dissolved oxygen 25-35%, and cultivation time 22h; the cultivation conditions for synergistic bacteria are: temperature 30℃±0.5℃, pH 5.5-6.0, humidity at the top of the fermenter 70-5%, dissolved oxygen 40-50%, and cultivation time 36h.

[0068] Step 3: Secondary premix tank culture: Take the seed liquid of the single strain obtained in Step 2, mix it with the seed liquid to be mixed in a certain proportion, keep it at 30℃ for 30 minutes to obtain the seed liquid of the mixed strain.

[0069] The second set of embodiments: Bio-mineral fertilizer that combines coal gangue activation and heavy metal remediation.

[0070] This set of embodiments provides a bio-mineral fertilizer prepared using the microbial liquid compound inoculant described in the first set of embodiments, which combines coal gangue activation and heavy metal remediation.

[0071] In a specific embodiment of the present invention, the method for preparing the bio-mineral fertilizer includes:

[0072] A1: The microbial liquid compound inoculant described in claim 1, coal gangue powder (80-100 mesh), wheat bran, soybean meal, and electrochemical low-hertz water (small molecule cluster water) are added to an intelligent mixing system. After uniform mixing, the mixture is conveyed to an intelligent solid-state fermentation system production line via an independently invented intelligent material distribution system (patent authorization announcement number CN 116553963B) for the first solid-state fermentation. The intelligent fermentation system regulates the temperature during fermentation through a temperature sensor and a linked intermittent micro-burst oxygen frequency: the initial stage temperature is 42-45℃, maintained for 22-24 hours; the peak stage temperature is 60-65℃, maintained for 46-48 hours; and the final stage temperature is 40-45℃, maintained for 22-24 hours. When the temperature remains at the final stage temperature without fluctuation for 24 consecutive hours, the material moisture content is ≤40%, the material color is dark brown, there is no odor, and the material is loose to the touch without clumping, it indicates the first solid-state fermentation. Solid-state fermentation is complete. In A1, the total amount of solid components—coal gangue powder, wheat bran, and soybean meal—is 100%, with the following proportions: 90-95% 80-mesh coal gangue powder, 3-8% wheat bran, and 4-10% soybean meal. The initial moisture content of the coal gangue powder is 5-9%, and the initial moisture content of both wheat bran and soybean meal is 8-12%. After mixing the solid components evenly, electrolytic low-hertz water is added and stirred thoroughly. Simultaneously, a microbial liquid compound inoculant is added during the stirring process, at a rate of 0.8-1.2% of the total solid substrate, ultimately bringing the total moisture content of the material to 50-60%.

[0073] A2: The material obtained from the first solid-state fermentation in A1 is rearranged into the aging and fermentation production line through an intelligent material distribution system for the second aging and fermentation: The material is naturally piled up, and combined with intelligent monitoring, the residual heat and residual microorganisms of the material are used to complete the deep decomposition, which lasts for 10-12 days. When the material naturally cools down to a moisture content of 15-29%, the color is uniformly dark brown, the texture is loose, it has an earthy smell without other odors, the pH is stable at 6.5-7.5, and the number of beneficial live bacteria is ≥200 million / g, the second aging and fermentation is completed.

[0074] Experimental Example

[0075] Experimental Example 1. Effects of Electrochemical Low-Hertz Water on the Number of Viable Cells at Different Stages of Fermentation

[0076] Experimental group: Microbial liquid compound inoculant prepared according to the method in the first group of examples.

[0077] Control group: The electrochemical low-hertz water in the first embodiment was replaced with sterilized water, and all other operations were the same as in the first embodiment.

[0078] The viable cell counts at each fermentation stage were measured in both the experimental and control groups. Statistical analysis was performed using ANOVA (α=0.05), with at least three replicates per group. The results are shown in Table 2. At the end of the first time step, the viable cell count in the experimental group was 41.2% higher than that in the control group; at the end of the second time step, the viable cell count in the experimental group was 37.0% higher than that in the control group; at the end of the third time step, the viable cell count in the experimental group was 33.3% higher than that in the control group; and after 3 months of storage at room temperature, the viable cell count in the experimental group was 71.4% higher than that in the control group. This indicates that electrochemical low-Hertz water can effectively improve fermentation efficiency.

[0079] Table 2. Effects and mechanisms of electrochemical low-Hertz water on viable bacterial count

[0080]

[0081] Experimental Example 2. Effects of Using Bio-mineral Fertilizer

[0082] 2.1 Sweet potato experiment in sandy soil of Shijiazhuang, Hebei Province

[0083] (1) Test fertilizers and fertilization methods:

[0084] Processing Group 1:

[0085] Fertilizer: The biomineral fertilizer 1 prepared according to the preparation method in the second set of examples has a microbial liquid compound inoculant addition amount of 0.8%.

[0086] Application method: 1-2 weeks before sweet potato transplanting, mix bio-mineral fertilizer 1 (150 kg / mu) and Luyuan commercial organic fertilizer (1500 kg / mu) evenly and apply in furrows. At transplanting, dip the roots in bio-nitrogen fertilizer (6 kg / mu). During the seedling stage, drip irrigate with bio-nitrogen fertilizer (8 kg / mu). During the fruit enlargement stage, apply bio-nitrogen fertilizer (18 kg / mu) and bio-mineral fertilizer 1 (80 kg / mu) in furrows.

[0087] Processing Group 2:

[0088] Fertilizer: Replace the electrochemical low-hertz water in the preparation method of the second group embodiment with ordinary sterilized water, and the other operations are exactly the same as in the second group embodiment to obtain biomineral fertilizer 2;

[0089] Application method: Same as treatment group 1.

[0090] Control group 1:

[0091] Fertilizer: Replace the microbial liquid compound inoculant in the preparation method of the second group of examples with a single nitrogen-fixing bacterium, Azotobacter chrysophagus, and the other operations are exactly the same as in the second group of examples.

[0092] Application method: Same as treatment group 1.

[0093] Control group 2:

[0094] Fertilizers: Sinochem Compound Fertilizer (10-20-20), Luyuan Commercial Organic Fertilizer, Sinochem High Nitrogen Compound Fertilizer (20-10-10), and Hualufeng High Potassium Compound Fertilizer;

[0095] Application method: Before transplanting sweet potatoes, mix Sinochem compound fertilizer (10-20-20) (80kg / mu) and Luyuan commercial organic fertilizer (1500kg / mu) and apply in furrows. During the seedling stage, apply Sinochem high nitrogen compound fertilizer (20-10-10) (15kg / mu) and Hualufeng high potassium compound fertilizer (25kg / mu) in furrows.

[0096] (2) Test location:

[0097] The soil is aeolian sandy soil with an organic matter content of 0.8%-1.0%, available phosphorus / potassium / silicon (mg / kg) of 7.2±0.5 / 85±4.3 / 20±1.8, and a soil bulk density of 1.52±0.05 g / cm³. 3 .

[0098] (3) Experimental design:

[0099] The randomized block design (3 mu per group, 3 replicates) was used. After transplanting, irrigation was carried out in small amounts and frequently, and weeding was done manually. Only control group 2 was sprayed with carbendazim (80g / mu) when root rot occurred, while the other groups were not treated. The planted variety was sweet potato "Jishu 98" (drought-resistant, tolerant of poor soil, and the main variety planted in sandy areas).

[0100] (4) Experimental results:

[0101] The experimental results are shown in Table 3 below: Compared with treatment group 2, control group 1, and control group 2, treatment group 1 showed significantly increased plant height, stem diameter, number of tubers per plant, yield per unit area, and dry matter content; the incidence of root rot was significantly reduced; and the soil was improved after treatment group 1: the available silicon in the soil was significantly increased, and the soil bulk density was significantly reduced. The bio-mineral fertilizer provided by this invention effectively promotes the growth and yield of sweet potatoes, significantly reduces the incidence of root rot, and improves the soil; under the same application method and dosage, the effect of treatment group 1 is significantly better than that of treatment group 2, control group 1, and control group 2.

[0102] Table 3. Results of the sweet potato planting experiment

[0103]

[0104] 2.2 Maize Experiment in Cd-Contaminated Areas of Shandong

[0105] (1) Test fertilizers and fertilization methods:

[0106] Processing Group 1:

[0107] Fertilizer: Biomineral fertilizer 1 prepared according to the preparation method in the second set of examples, with a liquid compound microbial agent addition amount of 6%.

[0108] Application method: Use Luyuan commercial organic fertilizer (300 kg / mu) as base fertilizer, mix biological nitrogen fertilizer with seeds (0.9 kg / mu), and sow biological mineral fertilizer 1 (80 kg / mu) with the seeds along with the seeder at the time of sowing. Spray biological mineral fertilizer 1 (5 kg / mu) and water mixture (biological mineral fertilizer 1: water = 1:30) at the large trumpet stage, and take the supernatant for spraying.

[0109] Processing Group 2:

[0110] Fertilizer: Replace the electrochemical low-hertz water in the preparation method of the second group embodiment with ordinary sterilized water, and the other operations are exactly the same as in the second group embodiment to obtain biomineral fertilizer 2;

[0111] Application method: Same as treatment group 1.

[0112] Control group 1:

[0113] Fertilizer: Replace the microbial liquid compound inoculant in the preparation method of the second group of examples with a single nitrogen-fixing bacterium, Azotobacter chrysophagus, and the other operations are exactly the same as in the second group of examples.

[0114] Application method: Same as treatment group 1.

[0115] Control group 2:

[0116] Fertilizers: Sinochem Compound Fertilizer (15-15-15), Luyuan Commercial Organic Fertilizer, Shikefeng High Nitrogen Compound Fertilizer (30-5-5);

[0117] Application method: Use Sinochem compound fertilizer (15-15-15) (50kg / mu) and Luyuan commercial organic fertilizer (300kg / mu) as base fertilizer, and use Shikefeng high nitrogen compound fertilizer (30-5-5) (25kg / mu) during the large trumpet stage.

[0118] (2) Test location:

[0119] The soil was alluvial, with a total Cd content of 1.0±0.12 mg / kg, available phosphorus / silicon (mg / kg) of 8.5±0.6 / 22±1.9, and a pH value of 6.9±0.2.

[0120] (3) Experimental design:

[0121] The randomized block design was used, with each group consisting of 1.5 acres and three replicates. The corn variety "Ludan 505" was planted, and field management was consistent across all groups.

[0122] (4) Experimental results:

[0123] The experimental results are shown in Table 4: Compared with treatment group 2, control group 1, and control group 2, treatment group 1 showed significantly increased plant height, leaf area index, number of aerial roots, and yield per unit area; significantly reduced the incidence of stalk rot; and significantly reduced Cd content in grains. Treatment group 1 also improved the soil, significantly increasing the available phosphorus content. The bio-mineral fertilizer provided by this invention effectively promotes maize growth and yield per unit area, significantly reduces the incidence of stalk rot, and improves the soil. Under the same application method and dosage, treatment group 1 was significantly more effective than treatment group 2, control group 1, and control group 2.

[0124] Table 4. Results of the maize planting trial

[0125]

[0126] 2.3 Wheat Experiment in Tanghe, Nanyang, Henan

[0127] (1) Test fertilizers and fertilization methods:

[0128] Processing Group 1:

[0129] Fertilizer: The biomineral fertilizer 1 prepared according to the preparation method in the second set of examples has a liquid compound microbial agent added at a rate of 10%.

[0130] Application methods: Mix biological nitrogen fertilizer with seeds (1.6 kg / mu), mix biological mineral fertilizer 1 (100 kg / mu) with sowing, apply biological nitrogen fertilizer (8 kg / mu) during the greening stage, spray biological nitrogen fertilizer (10 kg / mu) during the jointing stage, apply organic fertilizer 300 kg / mu as base fertilizer, and spray the supernatant of the mixture of biological nitrogen fertilizer and water (biological nitrogen fertilizer 1: water = 1:30).

[0131] Processing Group 2:

[0132] Fertilizer: Replace the electrochemical low-hertz water in the preparation method of the second group embodiment with ordinary sterilized water, and the other operations are exactly the same as in the second group embodiment to obtain biomineral fertilizer 2;

[0133] Application method: Same as treatment group 1.

[0134] Control group 1:

[0135] Fertilizer: Replace the microbial liquid compound inoculant in the preparation method of the second group of examples with a single nitrogen-fixing bacterium, Azotobacter chrysophagus, and the other operations are exactly the same as in the second group of examples.

[0136] Application method: Same as treatment group 1.

[0137] Control group 2:

[0138] Fertilizers: Sinochem Compound Fertilizer (15-15-15), Luyuan Commercial Organic Fertilizer, Shikefeng High Nitrogen Compound Fertilizer (30-5-5).

[0139] Application method: Use Sinochem compound fertilizer (15-15-15) (50kg / mu) and Luyuan commercial organic fertilizer (300kg / mu) as base fertilizer, and use Shikefeng high nitrogen compound fertilizer (30-5-5) (25kg / mu) during the large trumpet stage.

[0140] (2) Test location:

[0141] Yellow-brown soil, with an organic matter content of 1.2%-1.4% and an available phosphorus / silicon ratio (mg / kg) of 12.5±0.8 / 70±4.5.

[0142] (3) Experimental design:

[0143] A randomized block design was adopted, with each group having a planting area of ​​2 mu (approximately 0.33 hectares) and 3 replicates. The wheat variety planted was "Zhengmai 379" (a high-quality, strong gluten wheat, the main variety), and the field management of each group was consistent.

[0144] (4) Experimental results:

[0145] The experimental results are shown in Table 5: Compared with treatment group 2, control group 1, and control group 2, treatment group 1 showed significantly higher plant height, effective tiller number, yield per unit area, thousand-grain weight, and protein content; after treatment group 1, the soil was improved: the soil organic matter content was significantly increased. The bio-mineral fertilizer provided by this invention effectively promotes wheat growth and yield, and improves soil quality; under the same application method and dosage, the effect of treatment group 1 was significantly better than that of treatment group 2, control group 1, and control group 2.

[0146] Table 5. Results of the wheat planting trial

[0147]

[0148] In summary, the bio-mineral fertilizer prepared by electrochemical low-hertz water in this application achieves a resource utilization rate of over 80% for coal gangue, a reduction in chemical fertilizer use of over 80%, and a heavy metal remediation rate of over 75%. It can be promoted to sandy soils, coal gangue accumulation areas, and heavy metal-contaminated farmland in North China and the Huang-Huai-Hai Plain, and is suitable for grain and cash crops, providing technical support for the green and sustainable development of agriculture.

Claims

1. A microbial liquid composite agent that combines coal gangue activation and heavy metal remediation, characterized in that, The following functional strains were prepared using electrochemical low-Hertz water through a three-stage sequential inoculation and fermentation process: *Pseudomonas putida* (a restorative bacterium), *Azotobacter chrysogenum* (a nitrogen-fixing bacterium), *Azospirillum brasiliensis* (a growth-promoting bacterium), *Pseudomonas fluorescens* (a phosphate-solubilizing bacterium), *Bacillus subtilis* (a biocontrol bacterium), *Bacillus mucilaginosus* (a potassium-solubilizing bacterium), *Bacillus megaterium* (a silica-solubilizing bacterium), and *Trichoderma echinococcus* (a synergistic bacterium). The inoculation and fermentation preparation includes the following steps: S1: Add electrochemical low-Hertz water to the sterilized main fermenter, and add pre-mixed seed solutions of repair bacteria, nitrogen-fixing bacteria, and growth-promoting bacteria. Start stirring and adjust the fermentation conditions to the first-order fermentation conditions: pH=6.5-7.0, dissolved oxygen content of 20-30%, temperature of 30℃±0.5℃, humidity of the top space of the fermenter of 90%-92%, and fermentation time of 23-25 ​​hours; the molecular clusters of the electrochemical low-Hertz water are ≤6 molecules. S2: After the S1 stage, adjust to the second-sequence fermentation conditions, and add pre-mixed phosphate-solubilizing bacteria and biocontrol bacteria seed liquid to the main fermenter, and add coal gangue powder to the main fermenter, continue stirring, and adjust the fermentation conditions to the second-sequence fermentation: pH=6.8±0.2, dissolved oxygen 30-40%, temperature 30℃±0.5℃, humidity of the top space of the fermenter 91%-93%, fermentation time 17-19 hours; S3: After the fermentation of S2 stage is completed, the pre-mixed seed liquid of potassium-solubilizing bacteria, silica-solubilizing bacteria and synergistic bacteria is added to the main fermenter, and stirring is continued. The fermentation conditions are adjusted to the third time sequence: pH=7.0±0.2, dissolved oxygen content is 40-50%, temperature is 30℃±0.5℃, humidity of the top space of the fermenter is 85%-88%, and fermentation time is 35-37 hours. When the total viable bacteria count in the main fermenter is ≥2×10 10 Fermentation ends when CFU / mL; The volume ratio of the seed liquid of the repair bacteria, nitrogen-fixing bacteria, growth-promoting bacteria, phosphate-solubilizing bacteria, biocontrol bacteria, potassium-solubilizing bacteria, silica-solubilizing bacteria and synergistic bacteria is 2:3:1:1:1:1:1:1; During the S1 stage, when fermentation reaches the middle of the logarithmic growth phase, nutrient factors citric acid and sodium molybdate, along with a low concentration of Cr in the stress-inducing solution, are added. 6+ After fermentation begins in stage S2 and the pH of the system stabilizes, add glycerol and coal gangue powder as nutrients; when stage S3 is started, add potassium feldspar powder and humic acid as nutrients. S1 contains citric acid, sodium molybdate, and low concentrations of Cr. 6+ The treatment employs pulse dosing, with each addition being 1 / 3 of the total required amount, spaced 2 hours apart, for a total of 3 times. The pulse dosing is performed using a slow-flow injection method, completed within 10 minutes. One hour after each injection of citric acid and sodium molybdate, a low concentration of Cr is slowly injected. 6+ One-third of the total liquid demand must be completed within 5 minutes. In S2, glycerol and coal gangue powder are added simultaneously. The glycerol is continuously injected using a peristaltic pump, and the coal gangue powder is added in batches through a sterile crushing and feeding device. In S3, potassium feldspar powder and humic acid are added simultaneously. The potassium feldspar powder is added in batches through a sterile feeding device, while the humic acid is continuously injected through a peristaltic pump.

2. The microbial liquid composite agent based on claim 1, which combines coal gangue activation and heavy metal remediation, is characterized in that, The end of stage S1 is marked by a total bacterial count ≥ 1 × 10⁻⁶ in the main fermenter. 9 CFU / mL, heavy metal adsorption rate ≥60%, nitrogenase activity ≥20 nmol C2H4 / (mL·h), IAA content ≥10 mg / L; the end of the S2 phase is marked by: total bacterial count ≥1.5 × 10⁻⁶. 9 The CFU / mL concentration was measured, and the effective phosphorus concentration in the supernatant was ≥50 mg / L, with a Bacillus subtilis spore count ≥90%. The end of the S3 phase was marked by a total viable count ≥2 × 10⁻⁶. 10 The total number of viable bacteria is CFU / mL, with a potassium feldspar decomposition rate ≥35%, a silicon leaching rate ≥25%, and a coal gangue organic matter conversion rate ≥30%; the total number of viable bacteria includes fungal spores and bacteria.

3. The microbial liquid composite agent based on claim 1, which combines coal gangue activation and heavy metal remediation, is characterized in that... The preparation and mixing process of the seed solution described in S1-S3 includes the following steps: Step 1: Pretreatment: Each strain was inoculated into a dedicated culture medium and cultured at 30°C for 24-48 hours until the logarithmic phase. The remediation bacteria were cultured in LB medium with 0.01% cadmium ion concentration; the nitrogen-fixing bacteria were cultured in Assambe nitrogen-free medium; the phosphate-solubilizing bacteria were cultured in KB medium; the biocontrol bacteria were cultured in LB medium; the potassium-solubilizing bacteria were cultured in potassium-solubilizing medium; the silica-solubilizing bacteria were cultured in beef extract peptone medium with 2% silica mineral powder medium; the Trichoderma echinosporum was induced to culture in PDA medium containing 0.5% sand. The dedicated culture medium was prepared with electrolytic low-hertz water and sterilized at 121°C for 20 minutes. Step 2: Primary Seed Tank Cultivation: Configure independent primary seed tanks and add the strains cultivated in Step 1 to different independent primary fermenters. After cultivation for a certain period, obtain seed solutions for single strains. The cultivation conditions for repair bacteria and nitrogen-fixing bacteria are: temperature 30℃±0.5℃, humidity at the top of the fermenter 88-90%, dissolved oxygen 20-30%, and cultivation time 24 hours. The cultivation conditions for phosphate-solubilizing bacteria, potassium-solubilizing bacteria, and silica-solubilizing bacteria are: temperature 30℃±0.5℃, humidity at the top of the fermenter 90-92%, and dissolved oxygen 30-40%. The cultivation conditions for growth-promoting bacteria and biocontrol bacteria are: temperature 30℃±0.5℃, humidity at the top of the fermenter 85-88%, and dissolved oxygen 25-35%. The cultivation conditions for synergistic bacteria are: temperature 30℃±0.5℃, pH 5.5-6.0, humidity at the top of the fermenter 70-5%, and dissolved oxygen 40-50%. Step 3: Secondary premix tank culture: Take the seed liquid of the single strain obtained in Step 2, mix it with the seed liquid to be mixed in a certain proportion, keep it at 30℃ for 30 minutes to obtain the seed liquid of the mixed strain.

4. A bio-mineral fertilizer that combines coal gangue activation and heavy metal remediation, characterized in that, It is prepared using the microbial liquid composite agent that combines coal gangue activation and heavy metal treatment as described in any one of claims 1-3.

5. The bio-mineral fertilizer that combines coal gangue activation and heavy metal remediation according to claim 4, characterized in that, The method for preparing the bio-mineral fertilizer includes: A1: The aforementioned microbial liquid compound inoculant, 80-mesh coal gangue powder, wheat bran, soybean meal, and electrolytic low-hertz water are added to the mixing system. After the mixture is evenly stirred, it is conveyed to the solid-state fermentation system production line through an intelligent material distribution system for the first solid-state fermentation. The solid-state fermentation system automatically adjusts the temperature during the fermentation process through a temperature sensor linked to the intermittent micro-burst oxygen frequency: the initial stage temperature is 42-45℃, maintained for 22-24 hours; the peak stage temperature is 60-65℃, maintained for 46-48 hours; and the final stage temperature is 40-45℃, maintained for 22-24 hours. A2: The material obtained from the first solid-state fermentation in A1 is rearranged into the aging and fermentation production line for a second aging and fermentation through an intelligent material distribution system: the naturally piled material is deeply decomposed by utilizing the residual heat and residual microorganisms of the material, and the fermentation ends after 10-12 days.

6. The bio-mineral fertilizer that combines coal gangue activation and heavy metal remediation according to claim 5, characterized in that, The completion of the first solid-state fermentation in A1 is marked by the following: the temperature is maintained at the final stage temperature for 24 consecutive hours, the material humidity is ≤40%, the material color is dark brown, there is no odor, it is loose when squeezed, and there is no stickiness.

7. The bio-mineral fertilizer that combines coal gangue activation and heavy metal remediation according to claim 5, characterized in that, The completion of the second aging and fermentation in A2 is indicated by the following: the material naturally cools down to a moisture content of 15-29%, has a uniform dark brown color, a loose texture, an earthy smell without other odors, a pH of 6.5-7.5, and a beneficial viable bacteria count ≥200 million / g. In A1, the total amount of solid components—coal gangue powder, wheat bran, and soybean meal—is 100%, with the following proportions: 90-95% 80-mesh coal gangue powder, 3-8% wheat bran, and 4-10% soybean meal. The initial moisture content of the coal gangue powder is 5-9%, and the initial moisture content of the wheat bran and soybean meal is 8-12%. After the above solid components are mixed evenly, electrolytic low-hertz water is added and stirred thoroughly. Simultaneously, a microbial liquid compound inoculant is added during the stirring process, with the addition amount calculated as 0.8-1.2% of the total solid substrate, ultimately bringing the total moisture content of the material to 50-60%.

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