Micro-ecological restoration liquid complex microbial inoculant suitable for heavy metal contaminated soil and biological bacterial fertilizer of micro-ecological restoration liquid complex microbial inoculant

Abstract

The invention relates to the field of microbial agents, in particular to a micro-ecological restoration liquid composite microbial agent suitable for heavy metal contaminated soil and a biological bacterial fertilizer and a preparation method thereof. The micro-ecological restoration liquid complex microbial inoculant suitable for the heavy metal contaminated soil comprises nitrogen-fixing bacteria: azotobacter chroococcum ACCC 11104, phosphate solubilizing bacteria: pseudomonas fluorescens ACCC 03031, potassium solubilizing bacteria: bacillus mucilaginosus CCTCC KB20082790, growth-promoting bacteria: azotobacter braziliensis CCTCC AB 91001, bacillus subtilis CGMCC 17161, synergistic bacteria: trichoderma asperellum CGMCC 3.17461, and restoration bacteria: pseudomonas putida CGMCC 1.4533. The biological bacterial fertilizer for the heavy metal contaminated soil is prepared from the liquid complex microbial inoculant, compared with a conventional biological bacterial fertilizer, the adaptability to the heavy metal contaminated soil is greatly improved, the heavy metal contaminated soil is effectively improved, and the crop yield is remarkably increased.

Description

Technical Field

[0001] This invention relates to the field of microbial agents, specifically to a micro-ecological remediation liquid compound microbial agent and its bio-fertilizer suitable for heavy metal contaminated soil. Background Technology

[0002] Heavy metal contaminated soils are rich in heavy metal pollutants such as cadmium (Cd), lead (Pb), mercury (Hg), and arsenic (As) due to industrial and mining pollution or natural geographical background. These pollutants affect normal plant growth and accumulate in plants, leading to serious food safety problems. Furthermore, heavy metal-contaminated soils are characterized by soil acidification, nutrient depletion, and degradation of biological characteristics, severely reducing crop yields. However, based on profound considerations of resource availability, social stability, ecological risks, and economic costs, my country cannot abandon the cultivation of heavy metal-contaminated soils. First, contaminated farmland is an important component of the national strategy to uphold the 1.8 billion mu (120 million hectares) red line for arable land and ensure food security. In the context of dense population and rapid urbanization, high-quality arable land resources are extremely scarce, and large-scale abandonment will directly impact total grain production. The reality of having no land to replace makes every acre of land crucial. Second, land is the most basic means of production and social security for farmers. Forced abandonment not only means depriving them of their income and food supply but also risks causing widespread poverty and social instability due to the failure to properly address their livelihoods, creating "ecological refugees." Furthermore, from an ecological perspective, abandoning farmland is not a safe approach. Once land is left uncultivated and lacks crop cover, it will exacerbate wind and water erosion, causing pollutants to spread with dust and runoff, polluting the surrounding environment and amplifying ecological disasters. Simultaneously, naturally growing plants or crops spontaneously planted by residents on the wasteland may absorb heavy metals and enter the food chain through other pathways. Proper planting management can effectively control this risk. Finally, the technology for completely remediating contaminated soil is extremely difficult and costly, and large-scale application is financially unsustainable. Therefore, the state adopts a "risk management" strategy—ensuring the safe use of land contaminated with heavy metals through measures such as adjusting planting structures, selecting low-accumulation varieties, and employing agronomic passivation.

[0003] On the premise of safety, how to efficiently utilize heavy metal-contaminated soil to increase crop yield has become a current research hotspot. Applying conventional fertilizers to heavy metal-contaminated soil can lead to soil acidification, significantly activating various heavy metals and greatly increasing food safety risks. Inorganic salt ions in conventional fertilizers can also complex with heavy metal ions, and these complexes are more likely to accumulate in crops, increasing food safety hazards. In recent years, using microbial agents to improve the soil micro-ecological environment and reduce reliance on chemical fertilizers has become a research and application hotspot. Therefore, developing a microbial agent suitable for heavy metal-contaminated soil to improve soil properties and promote crop growth has become a current research focus. Although some existing microbial agents have been applied to dryland areas, their effects are still unsatisfactory, with many problems: contaminated soil itself is a highly toxic environment; exogenous agents are not only directly toxic by heavy metals but also face fierce competition for nutrients and space from more adaptable native microorganisms. Furthermore, the constraints of complex environmental conditions such as soil pH and humidity make it difficult for them to survive and reproduce effectively. Secondly, the function of single-strain microorganisms is often limited to treating only a few heavy metals, making them inadequate for dealing with common complex pollution in reality. Their remediation process is slow and their effects are highly unstable, greatly affected by environmental fluctuations. Furthermore, the metabolic activities of some strains may actually activate heavy metals, increasing their mobility and bioavailability, thus posing a risk of secondary pollution. These combined factors make the effectiveness of single or simply combined microbial agents extremely unstable and unpredictable, making them unsuitable for the characteristics of heavy metal-contaminated soils.

[0004] Compared to single-strain or simple combinations of microbial agents, compound microbial agents offer the core advantage of enhanced functionality, improved environmental adaptability, increased stability of effects, and more comprehensive functions through synergistic effects of the microbial community. However, separate fermentation of each microorganism in a compound microbial community leads to numerous production equipment and difficulties in production control. While mixed or partially mixed fermentation can reduce production equipment, various factors can cause problems. In an environment where multiple microorganisms coexist, some strains may grow too quickly, competing for resources and inhibiting the growth of other strains. This can result in the loss of the synergistic effect of the compound microbial community and even the production of undesirable metabolites such as odor substances, toxins, and pigments, affecting product quality and safety. Therefore, the industrial production of compound microbial agents faces significant challenges, easily leading to problems such as microbial community imbalance, functional failure or weakening, low yield, and difficulty in controlling product quality during large-scale industrial production. Existing technologies offer a range of solutions, such as optimizing the culture medium: adjusting the proportions of carbon sources, nitrogen sources, and trace elements to meet the growth needs of different strains and avoid excessive abundance of a single nutrient; optimizing fermentation conditions: controlling temperature, pH, stirring rate, and aeration to promote the overall growth of the complex microorganisms and inhibit the overgrowth of dominant strains; strain domestication: through multiple consecutive subculturing, allowing strains to adapt to specific fermentation environments and improving their tolerance and competitiveness; controlling the proportion of strains and inoculum size: optimizing the inoculum ratio of each strain to ensure the diversity and balance of the initial microbial community; and using selective inhibitors: adding low concentrations of selective inhibitors to the culture medium to inhibit the growth of dominant strains and maintain microbial community balance. However, these methods do not address how to better suit industrial production, thus employing relatively simplistic approaches that fail to comprehensively optimize various conditions while considering the complex characteristics of each strain. Furthermore, existing compound microbial agents suitable for arid lands suffer from unstable efficacy after fertilization, leading to short shelf life and high application rates. Summary of the Invention

[0005] To address the issues of low crop yields in heavy metal-contaminated soils due to high heavy metal ion content and poor soil quality, the ineffectiveness of conventional fertilizers which further deteriorate soil properties, and the difficulty in meeting the needs of heavy metal-contaminated soils with existing single or compound microbial agents, the industrial preparation of compound microbial agents is prone to problems such as microbial imbalance, low yield, and difficulty in controlling product quality to achieve optimal conditions. This invention provides a microecological remediation liquid compound microbial agent and its bio-fertilizer suitable for heavy metal-contaminated soils, solving the above problems.

[0006] The technical solution of the present invention is as follows:

[0007] This invention provides a microecological remediation liquid compound microbial agent suitable for heavy metal contaminated soil. Its characteristic is that it comprises the following seven functional bacterial strains, prepared using electrochemical low-Hertz water through a three-stage sequential inoculation and fermentation process: nitrogen-fixing bacteria: *Azotobacter chroococcum*, phosphate-solubilizing bacteria: *Pseudomonas fluorescens*, potassium-solubilizing bacteria: *Paenibacillus mucilaginosus*, growth-promoting bacteria: *Azospirillum brasilense*, biocontrol bacteria: *Bacillus subtilis*, synergistic bacteria: *Trichoderma asperellum*, and remediation bacteria: *Pseudomonas putida*.

[0008] S1: Propagation of nitrogen-fixing bacteria, growth-promoting bacteria and repair bacteria: Add electrolytic low-hertz water to the sterilized main fermenter, and add pre-mixed seed liquid of *Azotobacter chapensis*, *Azotobacter brasiliensis* and *Pseudomonas putida*. Adjust the fermentation conditions to the first-order fermentation conditions: pH = 7.0, dissolved oxygen 20-30%, temperature 30℃, humidity of the top space of the fermenter 92%±1%, fermentation time 25-27 hours;

[0009] S2: Introduce phosphate-solubilizing bacteria and biocontrol bacteria to relieve phosphorus limitation: After the S1 stage, adjust to the second sequential fermentation conditions, and add pre-mixed seed liquid of Pseudomonas fluorescens and Bacillus subtilis to the main fermenter. The second sequential fermentation conditions are: pH=6.5, dissolved oxygen 30-40%, temperature 30℃, humidity of the top space of the fermenter 92%±1%, fermentation time 19-21 hours;

[0010] S3: Initiate the synergistic metabolism of potassium-solubilizing bacteria and Trichoderma to activate mineral decomposition: After the fermentation of the S2 stage is completed, add Bacillus mucilaginosus seed liquid to the main fermenter. After fermentation for 12 hours, add Trichoderma echinococcosis seed liquid, turn on mechanical stirring and adjust to the third-sequence fermentation conditions: pH=5.5, dissolved oxygen 40-50%, temperature 28℃, humidity of the top space of the fermenter 88%±1%, fermentation for 37-39 hours;

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

[0012] Preferably, the ratio of repair bacteria: nitrogen-fixing bacteria: phosphorus-solubilizing bacteria: potassium-solubilizing bacteria: growth-promoting bacteria: biocontrol bacteria: synergistic bacteria is 2:3:1:1:2:1:1.

[0013] Preferably, the seed culture preparation method is as follows: seven functional bacterial strains are inoculated from the preservation slant onto a special slant culture medium and cultured at 30°C for 24-48 hours until the logarithmic growth phase (OD50). 600 When the concentration was ≥1.0, the samples were transferred to shake flasks for culture, and then transferred to seven primary seed tanks for independent culture.

[0014] The method for preparing the pre-mixed seed solution is as follows: when the bacterial count in the primary seed tank is ≥5×10⁻⁶... 8 At CFU / mL, premixed seed solutions of *Pseudomonas putida*, *Azotobacter chrysogenum*, and *Azotobacter brasiliensis* were mixed at a volume ratio of 2:3:2 to obtain premixed seed solution 1. Premixed seed solutions of *Pseudomonas fluorescens* and *Bacillus subtilis* were mixed at a volume ratio of 1:1 to obtain premixed seed solution 2. After incubation at 30°C for 30 minutes, the two premixed seed solutions were used for sequential fermentation.

[0015] When the number of Bacillus subtilis bacteria in the primary seed tank is ≥5×10 8 At CFU / mL, the number of Trichoderma acicularis spores is ≥1×10⁻⁶. 7 At a concentration of spores / mL, it can be used for sequential fermentation;

[0016] The nitrogen-fixing bacteria and growth-promoting bacteria were prepared using Asbe medium, the phosphate-solubilizing bacteria were prepared using KB medium, the biocontrol bacteria and repair bacteria were prepared using LB medium containing 5 mg / L cadmium, the potassium-solubilizing bacteria were prepared using potassium-solubilizing medium, and Trichoderma echinosporum was prepared using PDA medium; all five media were prepared using electrochemical low-hertz water and sterilized at 121°C for 20 minutes.

[0017] The inoculation amounts of the premixed seed solution 1, premixed seed solution 2, Bacillus mucilaginosus seed solution, and Trichoderma echinosporum seed solution are 11.8%-12.2%, 7.8%-8.2%, 2.8%-3.2%, and 1.8%-2.2% of the main fermentation tank volume, respectively. The inoculation amount of electrochemical low-hertz water is 74.4%-75.6% of the fermentation volume. The electrochemical low-hertz water has a molecular cluster of ≤6 molecules and a pH of 7.0–7.5.

[0018] Preferably, during the S1 stage, sodium citrate and sodium molybdate are added when fermentation reaches the middle of the logarithmic growth phase; after the S1 stage fermentation ends and the system pH stabilizes at 6.5±0.2, glycerol and calcium phosphate are added; when the S3 stage is started, potassium feldspar powder and humic acid are added.

[0019] Preferably, sodium citrate and sodium molybdate in S1 are added simultaneously and in batches using a pulsed slow-flow injection method. The concentration of sodium citrate is 0.5 g / L and the concentration of sodium molybdate is 10 μM. The sodium citrate is added in three batches, with each batch containing 1 / 3 of the total required amount, and the interval between batches is 2 hours.

[0020] The glycerol concentration in S2 is 5 mM, and it is added continuously at a flow rate of 0.5 g / L·h to avoid local concentrations exceeding 10 mM; the calcium phosphate concentration is 2 g / L, added in 3 portions, each portion being 1 / 3 of the total required amount, with an interval of 4 hours.

[0021] In S3, potassium feldspar powder and humic acid are added in two batches. The potassium feldspar powder has a particle size of 200 mesh and a concentration of 10 g / L. The humic acid is added after being dissolved in small molecular cluster water and has a concentration of 0.8 g / L. The two additions are 6 hours apart.

[0022] This invention also provides a micro-ecological remediation bio-fertilizer suitable for heavy metal contaminated soil, which is prepared using the above-mentioned micro-ecological remediation liquid compound microbial agent suitable for heavy metal contaminated soil.

[0023] Preferably, the preparation method includes:

[0024] A1: The above-mentioned liquid compound microbial agent, electrochemical low-hertz water, and solid components: lignite powder, wheat bran, and soybean meal are added to the mixing and stirring system. After being mixed evenly, the mixture is conveyed to the solid-state fermentation system production line through an intelligent material distribution system (patent authorization announcement number CN 116553963B) 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 through an intelligent material distribution system for a second aging and fermentation: the naturally piled material is deeply decomposed using the residual heat and residual microorganisms. After 10-12 days, the fermentation ends, and the micro-ecological remediation bio-fertilizer suitable for heavy metal contaminated soil is obtained.

[0026] Preferably, the total amount of solid components in A1—lignite powder, wheat bran, and soybean meal—is 100%, with the following proportions: lignite powder 70-85%, wheat bran 8-15%, and soybean meal 8-20%. The lignite powder has a particle size of 80-100 mesh and an initial moisture content of 10-15%, while the initial moisture content of the wheat bran and soybean meal is 8-12%. After the solid components are mixed evenly, electrochemical low-hertz water is added and stirred thoroughly. Simultaneously, a liquid compound microbial agent is added during the stirring process, with the addition amount calculated at 0.8-10% of the total solid components, so that the total moisture content of the material reaches 50-60%.

[0027] Preferably, the sign that the first fermentation of stage A1 has ended is: 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 sticking.

[0028] Preferably, the second fermentation in stage A2 is marked as follows: when 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 live bacteria count ≥200 million / g, the second fermentation is complete.

[0029] The beneficial effects of this invention are as follows:

[0030] This invention provides a microecological remediation liquid compound microbial agent suitable for heavy metal contaminated soil. Through a design of grouped and sequential fermentation in a main tank, it organically combines strains with different fermentation conditions that are mutually generative within the same timeframe and metabolically complementary between different timeframes. By designing the fermentation conditions for each timeframe, it ensures that seven functional bacteria can reproduce normally and perform their respective functions, solving the problem of low crop yields caused by high heavy metal ion content and soil infertility in heavy metal contaminated soil. *Pseudomonas fluorescens* and *Trichoderma echinococcosis* convert available heavy metals into stable states through organic acid secretion and enzymatic reduction, rapidly reducing environmental toxicity. *Bacillus subtilis* produces antibacterial substances that inhibit the reproduction of heavy metal-resistant pathogens, restoring the health of the soil microbial community and achieving passivation of heavy metal ions. *Bacillus mucilaginosus* and *Bacillus megaterium*, while adsorbing heavy metals, secrete polysaccharides to rebuild the aggregate structure, improve soil porosity and water and fertilizer retention capacity, solving the problem of soil infertility. *Azotobacter chrysogenum* and *Azospirillum brasiliensis* continuously fix nitrogen and provide growth hormones, promoting normal crop growth in contaminated soil, achieving simultaneous remediation and production. By designing a main tank for grouped and sequential fermentation, seven functional bacteria are cultured together. This allows the liquid compound microbial agent to not only have basic growth-promoting functions but also the function of remediating heavy metal ion pollution. It has high adaptability to heavy metal-contaminated soil. This solves the problem that in industrial-scale preparation of liquid compound microbial agents, the growth of Pseudomonas malodorosa (remediation bacteria) is inhibited by competition from Bacillus subtilis and Pseudomonas fluorescens, resulting in a severe reduction in its reproduction and failure of its remediation function. Other functional bacteria also fail to function due to nutrient competition and mutual inhibition of metabolites.

[0031] This invention groups seven functional bacteria according to their growth requirements and performs time-sequential culture:

[0032] The first inoculation sequence consists of *Azotobacter chrysogenum*, *Azospirillum brasiliensis*, and *Pseudomonas putida*, all Gram-negative bacteria with nitrogen-fixing and plant growth-promoting functions, suitable for synergistic growth in a neutral microaerobic environment. This invention sets the fermentation conditions for this sequence as follows: dissolved oxygen 20–30%, satisfying the low-oxygen environment required for nitrogenase activity while also considering the basal respiration and energy metabolism needs of the bacteria; pH 7.0, providing the optimal acid-base range for the efficient nitrogen fixation of *Azotobacter chrysogenum* and *Azospirillum brasiliensis*, while also facilitating the synthesis of ferrophiles and growth hormones by *Pseudomonas putida*; constant temperature fermentation at 30°C for 25–27 hours, coordinating the growth cycle and metabolic rate of the three bacteria, avoiding nitrogenase inactivation or premature bacterial death caused by high temperatures; and a humidity level of 92% ± 1% in the top space of the fermenter, creating a stable high-humidity microenvironment for early rapid proliferation and functional expression of the bacterial community, preventing delayed cell metabolism or hindered functional protein synthesis due to water evaporation stress. The three-strain co-fermentation results in significant functional complementarity: *Azotobacter chrysogenum* and *Azospirillum brasiliensis* jointly fix nitrogen, providing a continuous supply of soluble organic nitrogen to the system and directly supporting *Pseudomonas putida* in synthesizing growth-promoting substances; the plant growth hormones and siderophores produced by *Pseudomonas putida*, in turn, promote the biomass and nitrogen fixation efficiency of the two nitrogen-fixing bacteria. This time sequence constructs a nitrogen fixation, secretion, and growth-promoting cycle system through a simultaneous fermentation strategy, which not only simplifies the process but also strengthens microbial interactions and metabolic synergy, providing a sufficient nitrogen source and bioactive substance basis for subsequent inoculation of the complex microbial community. Changing the time sequence parameters or fermentation order will disrupt the dynamic balance between nitrogen fixation and nitrogen utilization, leading to ammonium ion accumulation that inhibits nitrogenase activity or causes carbon and nitrogen metabolism disorders.

[0033] The second inoculation sequence includes *Pseudomonas fluorescens* and *Bacillus subtilis*, both fast-growing bacteria that prefer a slightly acidic environment and have high oxygen consumption during metabolism (especially during spore formation). This invention sets the fermentation conditions for the second inoculation sequence as follows: dissolved oxygen 30-40% to support the high oxygen consumption metabolism of both bacteria; pH 6.5 to optimize enzyme activity and product synthesis; and short-term fermentation at 32°C to allow both bacteria to accelerate proliferation at a higher temperature. The humidity at the top of the fermenter is maintained at 92% ± 1%, precisely matching the stringent humidity requirements of *Pseudomonas fluorescens* during phosphatase secretion and *Bacillus subtilis* during the large-scale synthesis of lipopeptide antibiotics. This ensures the continuity of efficient synthesis and secretion of key metabolites during this stage and avoids changes in cell membrane permeability and extracellular enzyme inactivation caused by humidity fluctuations. In addition, both fermentation processes rely on the nitrogen source produced by the first fermentation to synthesize metabolic enzymes, which can make efficient use of the nitrogen source in the early stage. Furthermore, the activated phosphorus produced by the fermentation processes can provide nutrition for the third fermentation strain. If this stage is advanced, the utilization rate of phosphorus will decrease significantly.

[0034] The third time sequence employs a "step-by-step inoculation" strategy: first, *Bacillus mucilaginosus* seed culture is inoculated into the main fermenter, and then *Trichoderma echinococcus* seed culture is inoculated 12 hours later. *Bacillus mucilaginosus* is a typical phosphate-solubilizing and potassium-solubilizing bacterium, possessing both acid resistance and heavy metal tolerance. It can decompose heavy metal-bound minerals in the soil by secreting organic acids and specific enzyme systems. *Trichoderma echinococcus* is a strictly aerobic fungus, requiring a high-oxygen environment for sporulation, and its growth rate is significantly lower than that of *Bacillus mucilaginosus*. This invention, through a 12-hour delay in inoculation of *Trichoderma echinococcus*, effectively avoids the competitive inhibition of rapid bacterial proliferation on the initial growth of the fungus. Simultaneously, the chitinase secreted by *Trichoderma echinococcus* can regulate the population density of potential contaminants (such as pathogenic fungi) with chitinous cell walls in the fermentation system, maintaining the stability of the functional microbial community structure and indirectly enhancing the heavy metal mineral decomposition efficiency of *Bacillus mucilaginosus* (reducing competition from contaminants for nutrients and living space). The fermentation parameters at this stage were precisely set according to the principle of "synergistic metabolism of two strains and adaptation to heavy metal stress": the dissolved oxygen concentration was controlled at 60%, which not only fully met the strict aerobic sporulation and cellulase synthesis requirements of *Trichoderma echinococcosis*, but also adapted to the oxygen supply for the aerobic metabolism and heavy metal-bound mineral decomposition process of *Bacillus mucilaginosus*; the pH value was adjusted to 5.5, which not only met the optimal acidic environment for enzyme production of *Trichoderma echinococcosis*, but also was compatible with the acid-resistant characteristics of *Bacillus mucilaginosus*, while the weakly acidic environment could reduce the toxic stress of free heavy metal ions in the fermentation system and ensure the activity of functional strains; the fermentation temperature was maintained at 28℃, which could simultaneously meet the optimal growth requirements of both functional strains, promote the stable expression of their heavy metal tolerance-related functional genes (such as metal ion transporter genes and chelate peptide synthesis genes), and lay the foundation for the adsorption, fixation or activation of heavy metals in subsequent soil remediation. The humidity at the top of the fermenter is controlled at 88%±1%. While enhancing oxygen supply efficiency through mechanical stirring, this moderate humidity environment can precisely adapt to the functional metabolic needs of the two strains: on the one hand, it conforms to the metabolic characteristics of Bacillus mucilaginosus in decomposing heavy metal-bound minerals by secreting extracellular polysaccharides. Extracellular polysaccharides can form complexes with heavy metal ions, improving the passivation efficiency of heavy metals during mineral decomposition; on the other hand, it adapts to the life cycle needs of Trichoderma echinosporum in forming conidia and synthesizing cellulase, promoting robust mycelial growth. The developed mycelial network not only enhances the colonization ability of the inoculant in heavy metal-contaminated soil (by adsorbing heavy metal ions through mycelials, reducing bacterial toxicity stress), but also works synergistically with Bacillus mucilaginosus to construct a "bacterial-fungal" joint remediation system, giving full play to the synergistic function of mineral activation and heavy metal passivation / degradation, while avoiding excessive mycelial growth and metabolic shift due to excessive humidity (such as excessive consumption of nutrients for mycelial growth rather than the synthesis of heavy metal tolerance-related metabolites).

[0035] Specifically, this invention, through grouping and setting three time-differentiated fermentation conditions, ensures that there is no interspecies antagonism during the fermentation process of each strain, that its own propagation is not inhibited, and that the fermentation products of strains that propagate earlier do not adversely affect strains that propagate later, allowing them to be efficiently utilized by strains that propagate later, thus promoting their growth. It achieves low-oxygen protection for nitrogen-fixing bacteria and high-oxygen protection for Trichoderma; the pH is set according to the functional differentiation of the bacterial community to meet the requirements of nitrogen-fixing bacteria for a neutral environment and phosphate-solubilizing bacteria for an acidic environment. This invention uses a single independent main fermenter, with each group undergoing different time-series fermentation in parallel, avoiding idle tanks and reducing the probability of cross-contamination and production costs. Furthermore, due to the rational design, the secondary propagation process of the liquid composite bacterial agent (i.e., the time-series propagation in the main fermenter) only requires the addition of trace amounts of growth factors and electrolytic low-Hertz water, eliminating the need for additional carbon and nitrogen sources, simplifying the production process and effectively reducing production costs.

[0036] In the preparation of the liquid compound microbial agent, electrochemical low-Hertz water is used for strain activation, primary culture, and time-sequential culture in the main fermenter. This electrochemical low-Hertz water consists of six water molecules, 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, electrochemical low-Hertz water can also enhance the permeability of microbial cell membranes, promote the transmission of intercellular signaling molecules (such as quorum sensing substances), and optimize the metabolic network of the compound 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. In phosphate-solubilizing bacteria fermentation, electrochemical low-Hertz water can stabilize the pH of the fermentation broth at 6.5-7.0, promoting an increase in acid phosphatase activity of more than 40%, thereby significantly improving phosphorus solubility. Furthermore, electrochemically treated low-Hertz water promotes the reproduction and nitrogenase activity of *Azotobacter chamaescoparia* by providing highly active carbon sources (such as small-molecule organic carbon liquids) and optimizing the carbon-to-nitrogen ratio, thus establishing a nitrogen source foundation. It also optimizes the metabolic coordination of *Pseudomonas putida* in the complex system. This weakly alkaline water (pH 7.5–8.0) neutralizes organic acids (such as lactic acid and acetic acid) produced during fermentation, stabilizing the extracellular pH environment and ensuring optimal activity of enzymes related to the synthesis of hematoxylins and plant growth hormones. Additionally, it promotes the secretion of organic acids (such as gluconic acid and citric acid) and phytase by *Pseudomonas fluorescens*, dissolving insoluble phosphorus (such as calcium phosphate and apatite) and relieving phosphorus limitation. Furthermore, it enhances the adsorption and acidolysis capacity of *Bacillus mucilaginosus* for silicate minerals, releasing potassium minerals and stimulating mineral decomposition. Finally, it promotes plant growth by stimulating the secretion of plant hormones (such as IAA and gibberellin) and siderophores by *Azotobacter brasiliensis*. The advantages of electrochemically treated low-Hertz water for microbial communities are not only reflected in the performance improvement of individual strains, but also in the optimization of synergy among the microbial community: electrochemically treated low-Hertz water promotes the exchange of nutrients among different strains (e.g., nitrogen-fixing bacteria provide nitrogen sources, and phosphate-solubilizing bacteria provide phosphorus sources), 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 of the microbial community, and reduce metabolic inhibition; it enhances stress resistance, strengthens cell membrane stability, and improves the survival rate of liquid microbial agents under stress conditions such as high temperature and high salt. Therefore, small molecular clusters have unique physicochemical advantages in microbial fermentation, and can significantly improve microbial metabolic efficiency and product synthesis capabilities.

[0037] This liquid compound microbial agent, through the scientific formulation of seven functional bacteria, achieves a synergistic effect of soil remediation and nutrient enhancement for heavy metal contaminated soil. The systemic formulation of these seven functional bacteria constructs a multi-dimensional remediation system for heavy metal contaminated soil. Firstly, it utilizes a scientific formulation mechanism based on the adsorption, transformation, and stabilization of heavy metals: the extracellular polysaccharides and biocolloids secreted by *Bacillus mucilaginosus* and *Bacillus megaterium* effectively adsorb Pb. 2+ Cd2+ Heavy metal ions are reduced in bioavailability through complexation; surfactants and other substances produced by Bacillus subtilis can emulsify heavy metal pollutants and promote their accumulation on microbial surfaces; Pseudomonas fluorescens secretes organic acids to dissolve some heavy metal salts and uses reductases to convert Cr... 6+ High-valence metals with similar toxicity are reduced to lower valence states, significantly reducing their migration and toxicity. *Trichoderma echinosporum* efficiently adsorbs heavy metals through its hyphal network; its secreted chitinases and metallothioneins further integrate metal ions, forming a "hyphae-gel" complex barrier with the bacteria, blocking the transfer of heavy metals to crops. Nitrogen-fixing bacteria (*Azotobacter blazei*, *Azospirillum brasiliensis*) provide nitrogen nutrition, and their cell polysaccharides and extracellular polymeric substances (EPS) also participate in heavy metal fixation, jointly maintaining the safety of the rhizosphere microenvironment. Secondly, the fertility of polluted soil is continuously protected through a synergistic activation mechanism of nutrients: Azotobacter globosum and Azospirillum brasiliensis continuously fix nitrogen, providing crops with a stable source of ammonium nitrogen and compensating for nitrogen cycle disorders caused by pollution; Pseudomonas fluorescens secretes organic acids such as citric acid and oxalic acid, which activate and hold phosphorus while integrating heavy metal ions, reducing the dual stress of phosphorus fixation and heavy metal toxicity; Bacillus mucilaginosus decomposes silicate minerals to release potassium, and uses the generated organic acids to synergistically enhance the availability of phosphorus and potassium and the efficiency of pollution passivation with Pseudomonas fluorescens, breaking the vicious cycle of nutrients in heavy metal polluted land. Third, soil health is restored through the synergistic mechanism of microecological regulation and biocontrol: Bacillus subtilis produces lipopeptide antibacterial substances (such as surfactants) to inhibit the reproduction of heavy metal resistant pathogens, and its biofilm structure provides colonization protection for functional flora, enhancing the flora's resistance to stress; Trichoderma lyses the cell wall of pathogenic fungi through hyperparasitism, and the chitinase secreted simultaneously degrades residual organic pollutants, and synergistically inhibits soil-borne diseases with Bacillus subtilis; Trichoderma hyphal network not only expands the rhizosphere colonization range of functional bacteria, but also improves the structure of polluted soil microbial communities, enhances biodiversity and ecosystem stability.

[0038] Regarding the stability of the microbial agent, this agent ensures its functional continuity under heavy metal stress through multiple tolerance and self-protection mechanisms: the hyphae of *Trichoderma echinococcosis* and the biocollagen produced by *Bacillus mucilaginosus* jointly form a spatial network structure, encapsulating the functional microbial community and effectively buffering the impact of heavy metal ions; the antimicrobial peptides produced by *Bacillus subtilis* not only inhibit native bacteria but also enhance the collective resistance of the microbial community to heavy metals; *Pseudomonas fluorescens* possesses an oxidative stress tolerance mechanism, enabling it to maintain metabolic activity in heavy metal-contaminated environments. This symbiotic protection system significantly improves the survival rate and colonization ability of the microbial agent in contaminated soil, making the microbial remediation function more durable and reliable.

[0039] Regarding the application rate of this microbial agent, it exhibits high efficiency and low consumption. The functional microbial community can synergistically colonize and expand spatially: *Trichoderma echinosporum* acts as a mycelial carrier, carrying nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and heavy metal adsorbing bacteria that extend along soil pores, significantly improving the spatial distribution uniformity and contact efficiency of the agent; the biofilm formed by *Bacillus megaterium* and *Bacillus mucilaginosus* can encapsulate heavy metals and slowly release functional bacteria, achieving a long-lasting effect of the remediation factors; *Azospirillum brasiliensis* promotes crop root development, expands the rhizosphere remediation zone, and provides a broader colonization interface for the microbial community. This multi-faceted synergistic and microecological construction strategy significantly improves the remediation efficiency per unit of microbial agent, thereby greatly reducing the application rate per acre, making it particularly suitable for low-cost remediation of medium to large areas of heavy metal-contaminated farmland.

[0040] From the formulation design of the liquid compound microbial agent, the proportion of remediation bacteria (Pseudomonas putida) is 2 parts. As the core of heavy metal pollution remediation, it effectively fixes Pb2 in the soil through its efficient adsorption and transformation capabilities. + Cd 2+ Heavy metal ions are reduced in bioavailability; nitrogen-fixing bacteria (Azotobacter chrysogenum) accounted for the highest proportion (3 samples), serving as the nitrogen supply center and continuously providing organic nitrogen sources for crops and functional microbial communities under pollution stress, alleviating nitrogen metabolism inhibition caused by heavy metals; growth-promoting bacteria (Azospirillum brasiliensis) accounted for 2 samples, stimulating crop root development by secreting growth hormones such as indoleacetic acid (IAA), enhancing their tolerance to heavy metal stress and nutrient absorption capacity; phosphate-solubilizing bacteria (Pseudomonas fluorescens) and potassium-solubilizing bacteria (Bacillus mucilaginosus) each accounted for 1 sample, activating fixed phosphorus and potassium in the soil while secreting organic acids to assist in the passivation of heavy metals, improving the synergistic efficiency of phosphorus and potassium elements and pollution remediation; biocontrol bacteria (Bacillus subtilis) and synergistic bacteria (Trichoderma echinosporum) each accounted for 1 sample, the former secreting antimicrobial peptides to inhibit pathogens and enhance the stress resistance of the microbial community, while the latter expanding colonization space, degrading organic pollutants, and promoting heavy metal stabilization through hyphal networks. The ratio of the two was controlled at a low level to maintain the ecological balance of the microbial community and avoid interspecific competition.

[0041] This invention constructs a functional system encompassing remediation, nitrogen fixation, phosphorus solubilization, potassium solubilization, growth promotion, biocontrol, and synergistic effects through the precise formulation and synergistic fermentation of seven types of functional bacteria. This system perfectly addresses the core challenges of heavy metal-contaminated soils, including high toxicity, low fertility, and microecological imbalance. A high proportion of remediation and nitrogen-fixing bacteria effectively passivates heavy metals and replenishes nitrogen, addressing the combined stresses of pollution and nutrient deficiency. Growth-promoting and phosphorus- and potassium-solubilizing bacteria synergistically enhance crop tolerance and rhizosphere nutrient activation efficiency, breaking the vicious cycle of nutrient depletion in polluted areas. Low-volume formulations of biocontrol and synergistic bacteria significantly improve the environmental adaptability and ecological stability of the microbial agent without interfering with the remediation function of the main microbial community. This formulation design further incorporates an electrochemical low-hertz water fermentation process, significantly improving the yield and activity of the microbial agent and ensuring the survival rate and functional continuity of the liquid compound microbial agent under high heavy metal pressure. Combined with industrial automated production control, this ultimately enables the bio-fertilizer to have a comprehensive advantage of strong adaptability to heavy metal contaminated soil, stable efficacy, and long-lasting remediation function, providing a reliable microbial solution for the safe agricultural use of heavy metal contaminated soil.

[0042] In the preparation of the liquid compound bacterial agent, succinic acid and sodium molybdate are added during the first sequential fermentation stage, reaching the mid-logarithmic growth phase (12-16 hours). This stage represents the peak metabolic activity of the bacteria and the optimal absorption efficiency of nutrients. Sodium citrate, as a precursor to heavy metal chelating agents, induces the synthesis of metal-chelating proteins by the repair bacteria. Sodium molybdate, as a core cofactor of nitrogenase, avoids metal toxicity due to its low concentration and maintains consistency with the concentration in the basal culture medium of nitrogen-fixing bacteria, thus helping to maintain nitrogenase stability. Simultaneously, it activates various molybdenum-dependent enzymes in Bacillus megaterium, thereby improving its nitrogen utilization efficiency and environmental adaptability. To prevent excessively high instantaneous concentrations from inhibiting bacterial growth, sodium molybdate is added in a pulsed manner.

[0043] After the first fermentation step is complete and the system pH stabilizes, the second fermentation step begins. At this stage, glycerol is added as a slow-release carbon source to induce phosphate-solubilizing bacteria to synthesize phosphatase. To avoid local concentrations exceeding 10 mM and inhibiting cell growth, glycerol is added continuously (flow rate 0.5 g / L·h). Calcium phosphate is added in three batches, utilizing its insoluble nature to continuously induce enzyme production by phosphate-solubilizing bacteria and preventing precipitation and uneven dissolved oxygen production caused by a single high-concentration addition. Furthermore, there is a synergistic effect between glycerol and calcium phosphate: glycerol provides energy, and calcium phosphate serves as a substrate; simultaneous addition of both can synergistically activate the phosphate-solubilizing metabolic pathway.

[0044] At the start of the third time step, add 200-mesh potassium feldspar powder (10 g / L) in two separate additions (6 hours apart) to prevent excessive addition from increasing the system viscosity and thus reducing dissolved oxygen efficiency. Oxalic acid secreted by *Trichoderma* can synergistically decompose the silicate structure in the potassium feldspar powder. The simultaneous addition of humic acid and potassium feldspar powder not only enhances the synergistic effect between *Trichoderma* and bacteria, but its carboxyl and phenolic hydroxyl groups can also complex heavy metals, improving remediation efficiency. Once the *Trichoderma echinococcus* seed culture is transferred to the fermenter, immediately start mechanical stirring (150-200 rpm) to increase the dissolved oxygen level to 40-50% (as *Trichoderma* is a strictly aerobic fungus), while simultaneously promoting full contact between the potassium feldspar powder and the fungal cells, improving the fungal reproduction efficiency.

[0045] This invention provides a micro-ecological remediation bio-fertilizer suitable for heavy metal contaminated soil. It uses liquid compound microbial agents, electrochemical low-Hertz water, and solid components—lignite powder, wheat bran, and soybean meal—as raw materials. While strictly ensuring organic matter >40%, effective viable bacteria count >200 million / gram, and fermentation raw material moisture content of 50%–60%, the use of wheat bran and soybean meal is reduced to the minimum level required for microbial nutrition, significantly increasing the proportion of lignite powder to 85%, thus drastically reducing raw material costs. This product employs a secondary solid-state fermentation process, combined with micro-burst oxygen technology to replace traditional mechanical turning, significantly reducing production energy consumption and labor costs. It also relies on an intelligent material distribution system to achieve fully unmanned continuous operation, making it particularly suitable for intensive, large-scale industrial production lines. Intermittent high-oxygen stimulation not only accelerates microbial proliferation and metabolism but also significantly enhances the activity and stability of the fermentation products. The micro-ecological remediation bio-fertilizer for heavy metal-contaminated soil provided by this invention uses a liquid compound microbial agent suitable for heavy metal-contaminated soil. This agent has multiple functions, including heavy metal adsorption and transformation, nitrogen fixation, phosphorus and potassium solubilization, growth promotion and stress resistance, and synergistic biocontrol. After being applied to the soil, it can quickly build a complete micro-ecological remediation system, specifically addressing prominent problems such as high soil toxicity, structural degradation, fertility depletion, and microbial imbalance under heavy metal pollution conditions. Compared with ordinary urea fertilizer and ordinary microbial fertilizer, the bio-fertilizer provided by this invention, relying on industrial fermentation design and the synergistic advantages of multifunctional microbial communities, shows significant high efficiency and stability in actual farmland applications: the application amount is reduced by 75.2-90.5% compared with urea for the same area, and it can reduce the Cd content of polluted soil by 75.8-78.1%, increase soil nitrogenase activity by 332.7%, increase root vitality by 70.8%-71.6%, reduce crop disease incidence by 72.0%, and increase yield by 20.5-22.8%. This bio-fertilizer successfully solves the problems of unstable efficacy, large application rates, and short duration of effect of traditional single or simple combination microbial agents in practical applications, providing a reliable technical solution for the green agricultural remediation and sustainable production of heavy metal contaminated soil. Detailed Implementation

[0046] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0047] Example 1: Preparation of a liquid compound microbial agent suitable for soil microecological remediation in arid land

[0048] The microecological remediation liquid compound bacterial agent for heavy metal contaminated soil described in this embodiment includes seven functional bacterial strains: remediation bacteria, nitrogen-fixing bacteria, phosphate-solubilizing bacteria, potassium-solubilizing bacteria, growth-promoting bacteria, biocontrol bacteria, and synergistic bacteria. The remediation bacteria are *Pseudomonas putida*, the nitrogen-fixing bacteria are *Azotobacter chroococcum*, the phosphate-solubilizing bacteria are *Pseudomonas fluorescens*, the potassium-solubilizing bacteria are *Paenibacillus mucilaginosus*, the growth-promoting bacteria are *Azospirillum brasilense*, the biocontrol bacteria are *Bacillus subtilis*, and the synergistic bacteria are *Trichoderma asperellum*. The total effective bacterial count is ≥2 × 10⁻⁶. 10 CFU / mL, fungal spore count ≥1×10 8 Spores / mL; The seven functional strains of *Pseudomonas putida*, *Azotobacter chrysogenum*, *Pseudomonas fluorescens*, *Bacillus mucilaginosus*, *Azotobacter brasiliensis*, and *Bacillus subtilis* were prepared by liquid fermentation using electrolytic low-Hertz water. The liquid fermentation specifically includes the following stages:

[0049] Seven bacterial strains were inoculated from preservation slant agar to specialized slant culture media: nitrogen-fixing bacteria on Assab medium, phosphate-solubilizing bacteria on KB medium, biocontrol bacteria on LB medium, potassium-solubilizing bacteria on potassium-solubilizing medium, *Trichoderma echinosporum* on PDA medium, and the remediation bacteria on LB medium containing 5 mg / L cadmium. All five media were prepared using electrochemically treated low-Hertz water. The cultures were incubated at 30°C for 24-48 hours until the logarithmic growth phase (OD50). 600 When the concentration was ≥1.0, the samples were transferred to shake flasks for culture, and then transferred to seven primary seed tanks for culture.

[0050] When the bacterial count is ≥5×10 8At a CFU / mL concentration, premixed seed solutions 1 were prepared by mixing *Pseudomonas putida*, *Azotobacter chrysogenum*, and *Azotobacter brasiliensis* seed solutions at a volume ratio of 2:3:2. Premixed seed solution 2 was prepared by mixing *Pseudomonas fluorescens* and *Bacillus subtilis* seed solutions at a volume ratio of 1:1. After incubation at 30°C for 30 minutes, both premixed solutions could be used for sequential fermentation. When the *Bacillus subtilis* bacterial count was ≥5×10⁻⁶, the results were obtained. 8 At CFU / mL, the number of Trichoderma acicularis spores is ≥1×10⁻⁶. 7 When the spore count is 1 mL, it can be used for sequential fermentation.

[0051] The inoculation amounts of the premixed seed solution 1, premixed seed solution 2, Bacillus mucilaginosus seed solution, and Trichoderma echinosporum seed solution are 11.8%-12.2%, 7.8%-8.2%, 2.8%-3.2%, and 1.8%-2.2% of the main fermentation tank volume, respectively. The inoculation amount of electrochemical low-hertz water is 74.4%-75.6% of the fermentation volume. The electrochemical low-hertz water has a molecular cluster of ≤6 molecules and a pH of 7.0–7.5.

[0052] The subsequent three-stage sequential culture was carried out in the main fermenter:

[0053] S1: Propagation of nitrogen-fixing bacteria, growth-promoting bacteria, and repair bacteria: Add electrolytic low-hertz water to the sterilized main fermenter, and add pre-mixed seed solutions of *Azotobacter chapensis*, *Azotobacter brasiliensis*, and *Pseudomonas putida* (premixed seed solution 1), with an inoculation volume of 10% of the fermentation volume; adjust to the first-order fermentation conditions: pH = 7.0, dissolved oxygen 20-30%, temperature 30℃, humidity of the fermenter top space 92%±1%, fermentation for 25-27 hours; when fermentation reaches the middle of the logarithmic growth phase, add growth factors sodium citrate and sodium molybdate. Sodium citrate and sodium molybdate are added simultaneously using a pulsed slow-flow injection method, with the concentration of sodium citrate being 0.5 g / L and the concentration of sodium molybdate being 10 μM. Each addition is 1 / 3 of the total required amount, with an interval of 2 hours, for a total of 3 times.

[0054] S2: Introduction of phosphate-solubilizing bacteria and biocontrol bacteria: After the S1 stage, adjust to the second fermentation conditions: pH 6.5, dissolved oxygen 30-40%, temperature 32℃, humidity 90%, and humidity of the top space of the fermenter 92%±1%. When the pH in the main fermenter stabilizes at 6.5±0.2, add the growth factors glycerol and calcium phosphate, and add a pre-mixed seed solution of *Pseudomonas fluorescens* and *Bacillus subtilis* (premixed seed solution 2) to the main fermenter at a rate of 5% of the fermentation volume. Ferment for 19-21 hours. Glycerol is added continuously at a concentration of 5 mM and a flow rate of 0.5 g / L·h to avoid local concentrations exceeding 10 mM. The calcium phosphate is added at a concentration of 2 g / L in three batches, each batch containing 1 / 3 of the total required amount, with an interval of 4 hours.

[0055] S3: Initiating the synergistic metabolism of potassium-solubilizing bacteria and Trichoderma to activate mineral decomposition: After the S2 stage fermentation, Bacillus mucilaginosus seed liquid is added to the main fermenter at an inoculation rate of 3% of the fermentation volume. The humidity at the top of the fermenter is 88%±1%. Then, potassium feldspar powder and humic acid are added simultaneously in two separate additions, 6 hours apart. The potassium feldspar powder has a particle size of 200 mesh and a concentration of 10 g / L. The humic acid is dissolved in small-molecule cluster water and added at a concentration of 0.8 g / L. After 12 hours of fermentation, Trichoderma echinococcus seed liquid is added at an inoculation rate of 2% of the fermentation volume. Mechanical stirring is started and the fermentation conditions are adjusted to the third-sequence fermentation conditions: pH 5.5, dissolved oxygen 60%, temperature 28℃, fermentation for 34-36 hours. After fermentation is complete and the pH of the system is stable, glycerol and calcium phosphate are added. The potassium feldspar powder has a particle size of 200 mesh and a concentration of 10 g / L, added 6 hours apart. When the viable bacteria count in the main fermenter is ≥2×10⁻⁶... 10 Fermentation ends when CFU / mL is reached, yielding the microbial compound agent suitable for heavy metal contaminated soil remediation.

[0056] In some embodiments of the present invention, the fermentation equipment used in the above fermentation process includes:

[0057] The main fermentation tank includes a three-layer inclined blade agitator, jacketed heating / cooling, three humidity sensors, a saturated humidifier, a refrigerated dehumidifier, a hot air circulation device, a condensate return and discharge system, an automated control system, and a functional detection module.

[0058] In some embodiments of the present invention, the main fermentation tank has a volume of 3000L, and the humidity sensors are respectively placed on the top / middle / bottom of the tank, spaced 15cm apart; the saturated humidifier has an adjustable water temperature of 30-32℃ and a processing capacity of 6m³. 3 / h; the refrigerated dehumidifier can cool down to 15℃, and the humidity after dehumidification is ≤80%; the hot air circulation device has an air temperature of 30℃±1℃ and an air speed of 0.5-1.2m / s, which can be adjusted as needed; the condensate return and discharge system is equipped with a 0.22μm sterile filter membrane; the automatic control system includes PLC linkage functions for humidity, temperature, dissolved oxygen, and stirring speed, with a response time of <10s, and can preset parameter curves for dryland farming; the functional detection module monitors the effective phosphorus / potassium concentration online and detects viable bacteria count, proline, ferrophilic acid, and nitrogenase activity offline.

[0059] In some embodiments of the present invention, the humidity of the space above the fermentation tank is regulated by the humidity sensor, the saturated humidifier, and the refrigerated dehumidifier.

[0060] Example 2: Preparation of a soil microecological restoration bio-fertilizer suitable for arid land

[0061] The bio-fertilizer described in this embodiment is prepared using the soil micro-ecological restoration liquid compound microbial agent suitable for arid land described in Example 1.

[0062] In a specific embodiment of the present invention, the raw materials of the bio-fertilizer include liquid compound microbial agent, electrochemical low-Hertz water, and solid components: lignite powder, wheat bran, and soybean meal. The total amount of the solid components is 100%, and the proportions of the three components are: lignite powder 70-85%, wheat bran 8-15%, and soybean meal 8-20%. The lignite powder has a particle size of 80-100 mesh and an initial moisture content of 10-15%. The initial moisture content of both 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. Simultaneously, the liquid compound microbial agent is added during the stirring process, with the addition amount calculated at 0.8-10% of the total solid components, ultimately bringing the total moisture content of the material to 50-60%.

[0063] The method for preparing the bio-fertilizer includes:

[0064] A1: The above-mentioned liquid compound microbial agent suitable for heavy metal contaminated soil, lignite powder, wheat bran, soybean meal, and electrochemical low-hertz water are added to the intelligent mixing system. After being mixed evenly, the mixture is conveyed to the intelligent solid-state fermentation system production line through the 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 the fermentation process through temperature sensors and linkage 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; the final stage temperature is 40-45℃, maintained for 22-24 hours. When the temperature does not rise or fall for 24 consecutive hours, the material moisture content is ≤40%, the material color is dark brown, there is no odor, it is loose when squeezed, and there is no stickiness, the fermentation of stage A1 ends.

[0065] A2: The material obtained from the first 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 fermentation is completed, and the micro-ecological remediation bio-fertilizer suitable for heavy metal contaminated soil is obtained.

[0066] Experiment Example 1: Effect of Electrochemical Low-Hertz Water on the Number of Viable Cells at Different Stages of Fermentation

[0067] Experimental group: Liquid compound microbial agent suitable for heavy metal contaminated soil, prepared according to the method of Example 1.

[0068] Control group: The electrochemical low-hertz water in Example 1 was replaced with sterilized water, and all other operations were the same as in Example 1.

[0069] The number of viable bacteria in the experimental and control groups at each fermentation stage was measured. Statistical analysis was performed using ANOVA (α = 0.05), with at least three replicates per group. The results are shown in Table 1. At the end of the first time step, the number of viable bacteria in the experimental group increased by 53% compared to the control group; at the end of the second time step, the number increased by 48%; and at the end of the third time step, the number increased by 41%. This indicates that electrochemical low-Hertz water can effectively improve fermentation efficiency.

[0070] Table 1. Viable bacterial count

[0071]

[0072] Experiment Example 2: The effect of using bio-fertilizer on soil contaminated with heavy metals.

[0073] 2.1 Wheat Experiment

[0074] (1) Test location:

[0075] Tangshan City, Hebei Province, alluvial soil, pH: 6.8±0.2, total Cd content: 1.2±0.15mg / kg, available Cd content: 0.45±0.05mg / kg, available nitrogen, phosphorus and potassium content: 65±4.2, 9.8±0.7 and 82±5.1mg / kg, respectively.

[0076] (2) Test fertilizers and fertilization methods:

[0077] Processing Group 1:

[0078] Fertilizer: Bio-fertilizer 1 was obtained according to the preparation method of Example 2, with the amount of liquid compound microbial agent added being 0.8%.

[0079] Application method: Use Luyuan commercial organic fertilizer (900 kg / mu) as base fertilizer, and use bio-fertilizer 1 for seed dressing (1.6 kg / mu); apply by irrigation during the greening stage (8 kg / mu); and spray by spraying during the jointing stage (10 kg / mu).

[0080] Processing Group 2:

[0081] Fertilizer: Replace the small molecule cluster water in the preparation method of Example 2 with ordinary sterilized water, and perform the other operations exactly the same as in Example 2 to obtain bio-fertilizer 2;

[0082] Application method: Same as treatment group 1.

[0083] Control group 1:

[0084] Fertilizer: The liquid compound bacterial agent in the preparation method of Example 2 was replaced with a single brown azotocin agent, and the other operations were exactly the same as in Example 2, to obtain a single nitrogen-fixing fertilizer.

[0085] Application method: Same as treatment group 1.

[0086] Control group 2:

[0087] Fertilizers: Sinochem Compound Fertilizer (18-22-8), Luyuan Commercial Organic Fertilizer, Sinochem Urea (≥46.4%)

[0088] Application method: Apply Sinochem compound fertilizer (18-22-8) (50 kg / mu) and Luyuan commercial organic fertilizer (organic matter ≥35%) (900 kg / mu) as base fertilizer; apply Sinochem urea (≥46.4%) (15 kg / mu) as top dressing during the greening stage; apply Sinochem urea (≥46.4%) (10 kg / mu) as top dressing during the jointing stage.

[0089] (3) Experimental design:

[0090] A randomized block design was adopted, with each group having a planting area of ​​1 mu (approximately 0.16 acres) and 3 replicates. Field management was consistent across groups. The wheat variety "Jimai 44" had moderate cadmium tolerance and was the main variety planted in the polluted area.

[0091] (4) Experimental results:

[0092] The experimental results are shown in Table 2 below: Compared with treatment group 2 and control group 1, treatment group 1 showed significantly increased effective tiller number, grain filling rate, thousand-grain weight, yield per unit area, and soil nitrogenase activity, while grain Cd content, available Cd in soil, and sheath blight incidence index were significantly reduced. This indicates that bio-fertilizer 1, which is the bio-fertilizer for heavy metal contaminated soil provided by this invention, is significantly more effective than bio-fertilizer 2 in treatment group 2 and nitrogen-fixing fertilizer in control group 1 under the same application method and dosage. It can effectively promote wheat growth, increase yield, and reduce Cd content in grains and soil, as well as sheath blight incidence index. Compared with the control group 2, treatment group 1 showed significantly increased effective tiller number, grain filling rate, thousand-grain weight, yield per unit area, and soil nitrogenase activity, while grain Cd content, available Cd in soil, and sheath blight incidence index were significantly reduced. This indicates that the amount of bio-fertilizer 1, i.e., the bio-fertilizer for heavy metal contaminated soil provided by this invention, is much lower than that of ordinary commercial fertilizers and organic fertilizers, has high applicability to arid land, and its growth-promoting and soil remediation effects are significantly better than those of ordinary commercial fertilizers and organic fertilizers.

[0093] Table 2 Results of wheat planting experiment

[0094]

[0095] 2.2 Rice Experiment 1

[0096] (1) Test location:

[0097] Xinghua City, Jiangsu Province: Paddy soil, pH: 6.5±0.3, total Cd content: 1.0±0.12 mg / kg, available Cd content: 0.38±0.04 mg / kg, available potassium: 85±5.3 mg / kg.

[0098] (2) Test fertilizers and fertilization methods:

[0099] Processing Group 1:

[0100] Fertilizer: The bio-fertilizer 1 prepared according to the preparation method of Example 2 has a liquid compound microbial agent added at a rate of 10%.

[0101] Application method: Use Jiangsu Shangguyuanwo commercial organic fertilizer (500 kg / mu) as base fertilizer; use bio-fertilizer 1 for seed dressing (1.5 kg / mu); apply bio-fertilizer 1 (6 kg / mu) during the tillering stage; spray bio-fertilizer 1 (9 kg / mu) during the booting stage; spray bio-fertilizer 1 (2 kg / mu) at the early grain-filling stage. Note: Choose a sunny evening for spraying, focusing on the underside of the leaves. If it rains within 6 hours after spraying, re-spray.

[0102] Processing Group 2:

[0103] Fertilizer: Replace the small molecule cluster water in the preparation method of Example 2 with ordinary sterilized water, and perform the other operations exactly the same as in Example 2 to obtain bio-fertilizer 2;

[0104] Application method: Same as treatment group 1.

[0105] Control group 1:

[0106] Fertilizer: The liquid compound bacterial agent in the preparation method of Example 2 was replaced with a single brown azotocin agent, and the other operations were exactly the same as in Example 2, to obtain a single nitrogen-fixing fertilizer.

[0107] Application method: Same as treatment group 1.

[0108] Control group 2:

[0109] Fertilizers: Sinochem Fertilizer (20-15-5), Sushang Guyuanwo Commercial Organic Fertilizer, Sinochem Urea (≥46.4%), Sinochem Potassium Chloride (containing 60% K2O), Stanley Potassium Dihydrogen Phosphate (P2O5 52%, K2O 34%)

[0110] Application method: Before transplanting, evenly spread 50 kg / mu of Sinochem fertilizer (20-15-5) and 500 kg / mu of Jiangsu Shangguyuanwo commercial organic fertilizer, and rotary till into the soil; during the transplanting period, spread 8 kg / mu of Sinochem urea (≥46.4%); during the tillering period, spread 12 kg / mu of Sinochem urea (containing 46% nitrogen) + 8 kg / mu of Sinochem potassium chloride (containing 60% K2O); during the booting period, spread 5 kg / mu of Sinochem urea (containing 46% nitrogen) + 10 kg / mu of Sinochem potassium chloride (containing 60% K2O); at the early grain filling stage, spray 2 kg / mu of Sinochem urea (containing 46% nitrogen) + 150 g / mu of Stanley potassium dihydrogen phosphate (P2O5 52%, K2O 34%).

[0111] (3) Experimental design:

[0112] A randomized block design was adopted, with each group having a planting area of ​​1.5 mu and 3 replicates. The rice variety planted was "Nanjing 5055" (a high-quality rice with moderate Cd accumulation), and the field management of each group was consistent.

[0113] (4) Test results

[0114] The experimental results are shown in Table 3 below: Compared with treatment group 2 and control group 1, treatment group 1 showed increased root activity, prolonged tillering peak period (days), increased stem thickness, increased chlorophyll SPAD value, increased yield per unit area, and decreased Cd content in brown rice. This indicates that bio-fertilizer 1, i.e., the bio-fertilizer provided by this invention suitable for heavy metal contaminated soil, is significantly more effective than bio-fertilizer 2 in treatment group 2 and nitrogen-fixing fertilizer in control group 1 under the same application method and dosage, and can effectively promote rice growth and reduce Cd content in brown rice. Compared with control group 2, treatment group 1 showed increased root activity, prolonged tillering peak period (days), increased stem thickness, increased chlorophyll SPAD value, increased yield per unit area, and decreased Cd content in brown rice. This indicates that the dosage of bio-fertilizer 1, i.e., the bio-fertilizer provided by this invention suitable for heavy metal contaminated soil, is much lower than that of ordinary commercial fertilizers and organic fertilizers, and its effect on promoting rice growth, reducing Cd content in brown rice, and adaptability to heavy metal contaminated soils is significantly better than that of ordinary commercial fertilizers and organic fertilizers.

[0115] Table 3 Results of Rice Experiment 1

[0116]

[0117] 2.3 Rice Experiment Two

[0118] (1) Experimental location: Xuancheng City, Anhui Province, gleyed paddy soil, pH: 6.2±0.2, total Cd content in soil: 1.1±0.13mg / kg, available Cd content in soil: 0.41±0.03mg / kg.

[0119] (2) Test fertilizers and fertilization methods:

[0120] Processing Group 1:

[0121] Fertilizer: The bio-fertilizer 1 prepared according to the preparation method of Example 2 has a liquid compound microbial agent addition amount of 6%.

[0122] Application method: Use Jiangsu Shangguyuanwo commercial organic fertilizer (500 kg / mu) as base fertilizer; mix seed with bio-fertilizer 1 (1.5 kg / mu); broadcast bio-fertilizer 1 (6 kg / mu) during tillering stage; spray bio-fertilizer 1 (9 kg / mu) during booting stage; spray bio-fertilizer 1 (2 kg / mu) at the early grain-filling stage. Note: Choose a sunny evening for spraying, focusing on the underside of the leaves. If it rains within 6 hours after spraying, re-spray.

[0123] Processing Group 2:

[0124] Fertilizer: Replace the small molecule cluster water in the preparation method of Example 2 with ordinary sterilized water, and perform the other operations exactly the same as in Example 2 to obtain bio-fertilizer 2;

[0125] Application method: Same as treatment group 1.

[0126] Control group 1:

[0127] Fertilizer: The liquid compound bacterial agent in the preparation method of Example 2 was replaced with a single brown azotocin agent, and the other operations were exactly the same as in Example 2, to obtain a single nitrogen-fixing fertilizer.

[0128] Application method: Same as treatment group 1.

[0129] Control group 2:

[0130] Fertilizers: Sinochem Fertilizer (20-15-5), Jiangsu Shangguyuanwo Commercial Organic Fertilizer, Sinochem Urea (≥46.4%), Sinochem Potassium Chloride (containing 60% K2O), Stanley Potassium Dihydrogen Phosphate (P2O5 52%, K2O 34%).

[0131] Application method: Before transplanting, evenly spread 50 kg / mu of Sinochem fertilizer (20-15-5) and 500 kg / mu of Jiangsu Shangguyuanwo commercial organic fertilizer, and then rotary till into the soil; during the transplanting period, spread 8 kg / mu of Sinochem urea (≥46.4%); during the tillering period, spread 12 kg / mu of Sinochem urea (containing 46% nitrogen) and 8 kg / mu of Sinochem potassium chloride (containing 60% K2O); during the booting period, spread 5 kg / mu of Sinochem urea (containing 46% nitrogen) and 10 kg / mu of Sinochem potassium chloride (containing 60% K2O); at the early grain-filling stage, spray 2 kg / mu of Sinochem urea (containing 46% nitrogen) and 150 g / mu of Stanley potassium dihydrogen phosphate (P2O5 52%, K2O 34%).

[0132] (3) Experimental design:

[0133] Randomized block design: 1.5 mu per group, 3 replicates, with consistent field management. Rice variety "Nanjing 5055" (high-quality rice with moderate Cd accumulation) was planted.

[0134] (4) Test results

[0135] The experimental results are shown in Table 4. Compared with treatment group 2 and control group 1, treatment group 1 showed increased root activity, prolonged tillering peak period (days), increased stem thickness, increased chlorophyll SPAD value, increased yield, and decreased Cd content in brown rice. This indicates that bio-fertilizer 1, the bio-fertilizer provided by this invention suitable for heavy metal contaminated soil, is significantly more effective than bio-fertilizer 2 in treatment group 2 and nitrogen-fixing fertilizer in control group 1 under the same application method and dosage, effectively promoting rice growth and reducing Cd content in brown rice. Compared with control group 2, treatment group 1 showed increased root activity, prolonged tillering peak period (days), increased stem thickness, increased chlorophyll SPAD value, increased yield, and decreased Cd content in brown rice. This indicates that the dosage of bio-fertilizer 1, the bio-fertilizer provided by this invention suitable for heavy metal contaminated soil, is much lower than that of ordinary commercial fertilizers and organic fertilizers. Its effect on promoting rice growth, reducing Cd content in brown rice, and adaptability to heavy metal contaminated soil are significantly better than ordinary commercial fertilizers and organic fertilizers.

[0136] Table 4 Results of Rice Experiment 2

[0137]

[0138] 3.1 Comparison of the effects of bio-fertilizers produced based on liquid compound microbial agents and single nitrogen-fixing microbial agents

[0139] This invention uses a combination of seven functional strains—*Pseudomonas putida* (repair), *Azotobacter chrysogenum* (nitrogen fixation), *Pseudomonas fluorescens* (phosphate solubilization), *Bacillus mucilaginosus* (potassium solubilization), *Azotobacter brasiliensis* (growth promotion), *Bacillus subtilis* (biocontrol), *Trichoderma echinosporum* (antagonism), and *Pseudomonas putida* (repair)—to prepare a bio-fertilizer (treatment group 1). Through the synergistic effect of "repair, nitrogen fixation, phosphorus solubilization, potassium solubilization, growth promotion, and biocontrol," the growth promotion effect is significantly better than that of a single nitrogen-fixing bacterium inoculant (control group 1).

[0140] In wheat trials: Compared to control group 1, treatment group 1 showed a 28.6% increase in effective tiller number and a 30.8% increase in grain-filling rate.

[0141] The thousand-grain weight increased by 11.2%, the yield per unit area increased by 18.3%, the Cd content in the grain decreased by 55.6%, the available Cd in the soil decreased by 46.4%, the sheath blight index decreased by 58.4%, and the nitrogenase activity increased by 161.6% (Table 2).

[0142] In the rice experiment 1: compared with the control group 1, treatment group 1 showed a 46.2% increase in root activity, a 4-day earlier peak tillering period, a 14.3% increase in stem thickness, a 20.9% increase in chlorophyll SPAD value, a 14.8% increase in yield per unit area, and a 129% decrease in brown rice Cd content (Table 3).

[0143] In the second rice experiment: root activity increased by 47.9%, tillering peak occurred 4 days earlier, stem thickness increased by 14.8%, chlorophyll SPAD value increased by 21.4%, yield increased by 14.2%, and brown rice Cd content decreased by 52.9% (Table 4).

[0144] 3.2 Comparison of the effects of electrochemical low-hertz water and ordinary sterilized water

[0145] This invention uses electrochemical low-hertz water with a half-width at half-maximum (WHM) of ≤100Hz to prepare bio-fertilizer 1 (treatment group 1). By improving the permeability of microbial cell membranes and enhancing the adhesion and diffusion of bacterial agents, it is significantly superior to bio-fertilizer 2 (treatment group 2) made with ordinary water.

[0146] In wheat trials: Compared to control group 1, treatment group 1 showed a 12.5% ​​increase in effective tiller number and a 13.3% increase in grain-filling rate.

[0147] The thousand-grain weight increased by 6.0%, the yield per unit area increased by 7.3%, the Cd content in the grain decreased by 33.3%, the available Cd in the soil decreased by 31.8%, the sheath blight index decreased by 33.3%, and the nitrogenase activity increased by 22.9% (Table 2).

[0148] In the rice experiment 1: compared with the control group 1, treatment group 1 showed an increase of 17.8% in root activity, 2 days earlier in the peak tillering period, 5.9% in stem thickness, 8.3% in chlorophyll SPAD value, 6.0% in yield per unit area, and 57.1% in brown rice Cd content (Table 3).

[0149] In the second rice experiment: root activity increased by 18.3%, tillering peak occurred 2 days earlier, stem thickness increased by 6.1%, chlorophyll SPAD value increased by 8.5%, yield increased by 5.8%, and brown rice Cd content decreased by 33.3% (Table 4).

[0150] 3.4 Summary

[0151] This invention quantified and verified the technical effects of increased yield, improved quality, reduced disease, and improved soil through three crop planting trials: Treatment group 1, specifically the bio-fertilizer 1 provided by this invention suitable for heavy metal-contaminated soil, showed significant growth-promoting effects in three growth-promoting trials on both wheat and rice, not only increasing yield and quality but also significantly improving soil (Table 5). This indicates that the bio-fertilizer is suitable for grain crops grown on heavy metal-contaminated soil, not only promoting growth and increasing yield but also effectively improving soil properties, reducing heavy metal content, enhancing root nitrogenase activity, and improving root vitality.

[0152] Table 5. Comparison of effects between treatment group 1 and control group 2

[0153]

[0154] In summary, the bio-fertilizer for heavy metal contaminated soil provided by this invention achieves functional complementarity and rhizosphere ecological regulation through seven functional bacteria, coupled with electrochemical low-Hertz water enhancement and solid-state fermentation technology. This results in increased yield and quality of wheat and rice in heavy metal contaminated soil, while reducing the content of heavy metal ions (Cd) in the soil. This aligns with the strategic requirements of "reducing fertilizer use and increasing efficiency, improving arable land quality, and promoting green agriculture."

Claims

1. A microbial compound microbial agent suitable for the remediation of heavy metal contaminated soil, characterized in that, The following seven functional strains were prepared using electrochemical low-hertz water, i.e., small molecule cluster water, through a three-stage sequential inoculation and fermentation process: nitrogen-fixing bacteria: Azotobacter chroococcum, phosphate-solubilizing bacteria: Pseudomonas fluorescens, potassium-solubilizing bacteria: Paenibacillus mucilaginosus, growth-promoting bacteria: Azospirillum brasilense, biocontrol bacteria: Bacillus subtilis, synergistic bacteria: Trichoderma asperellum, and repair bacteria: Pseudomonas putida. S1: Propagation of nitrogen-fixing bacteria, growth-promoting bacteria and repair bacteria: Add electrolytic low-hertz water to the sterilized main fermenter, and add pre-mixed seed liquid of *Azotobacter chapensis*, *Azotobacter brasiliensis* and *Pseudomonas putida*. Adjust the fermentation conditions to the first-order fermentation conditions: pH = 7.0, dissolved oxygen 20-30%, temperature 30℃, humidity of the top space of the fermenter 92%±1%, fermentation time 25-27 hours; S2: Introduce phosphate-solubilizing bacteria and biocontrol bacteria to relieve phosphorus limitation: After the S1 stage, adjust to the second sequential fermentation conditions, and add pre-mixed seed liquid of Pseudomonas fluorescens and Bacillus subtilis to the main fermenter. The second sequential fermentation conditions are: pH=6.5, dissolved oxygen 30-40%, temperature 30℃, humidity of the top space of the fermenter 92%±1%, fermentation time 19-21 hours; S3: Initiate the synergistic metabolism of potassium-solubilizing bacteria and Trichoderma to activate mineral decomposition: After the fermentation of the S2 stage is completed, add Bacillus mucilaginosus seed liquid to the main fermenter. After fermentation for 12 hours, add Trichoderma echinococcosis seed liquid, turn on mechanical stirring and adjust to the third-sequence fermentation conditions: pH=5.5, dissolved oxygen 40-50%, temperature 28℃, humidity of the top space of the fermenter 88%±1%, fermentation for 37-39 hours; When the number of viable bacteria in the main fermenter is ≥2×10 10 Fermentation ends when CFU / mL is reached.

2. The microbial ecological remediation liquid composite bacterial agent for heavy metal contaminated soil according to claim 1, characterized in that, The ratio of repair bacteria: nitrogen-fixing bacteria: phosphate-solubilizing bacteria: potassium-solubilizing bacteria: growth-promoting bacteria: biocontrol bacteria: synergistic bacteria is 2:3:1:1:2:1:

1.

3. The microbial ecological remediation liquid composite bacterial agent for heavy metal contaminated soil according to claim 2, characterized in that, The seed culture preparation method is as follows: seven functional bacterial strains are inoculated from the preservation slant into a special slant culture medium and cultured at 30℃ for 24-48 hours until the logarithmic phase, i.e., OD. 600 When the value is ≥1.0, the samples were transferred to shake flasks for culture, and then transferred to seven primary seed tanks for independent culture. The method for preparing the pre-mixed seed solution is as follows: when the bacterial count in the primary seed tank is ≥5×10⁻⁶... 8 At CFU / mL, premixed seed solutions of *Pseudomonas putida*, *Azotobacter chrysogenum*, and *Azotobacter brasiliensis* were mixed at a volume ratio of 2:3:2 to obtain premixed seed solution 1. Premixed seed solutions of *Pseudomonas fluorescens* and *Bacillus subtilis* were mixed at a volume ratio of 1:1 to obtain premixed seed solution 2. After incubation at 30°C for 30 minutes, the two premixed seed solutions were used for sequential fermentation. When the number of Bacillus subtilis bacteria in the primary seed tank is ≥5×10 8 At CFU / mL, the number of Trichoderma acicularis spores is ≥1×10⁻⁶. 7 At a concentration of spores / mL, it can be used for sequential fermentation; The nitrogen-fixing bacteria and growth-promoting bacteria were prepared using Asbe medium, the phosphate-solubilizing bacteria were prepared using KB medium, the biocontrol bacteria and repair bacteria were prepared using LB medium containing 5 mg / L cadmium, the potassium-solubilizing bacteria were prepared using potassium-solubilizing medium, and Trichoderma echinosporum was prepared using PDA medium; all five media were prepared using electrochemical low-hertz water and sterilized at 121°C for 20 minutes. The inoculation amounts of the premixed seed solution 1, premixed seed solution 2, Bacillus mucilaginosus seed solution, and Trichoderma echinosporum seed solution are 11.8%-12.2%, 7.8%-8.2%, 2.8%-3.2%, and 1.8%-2.2% of the main fermentation tank volume, respectively. The inoculation amount of electrochemical low-hertz water is 74.4%-75.6% of the fermentation volume. The electrochemical low-hertz water has a molecular cluster of ≤6 molecules and a pH of 7.0–7.

5.

4. The microbial ecological remediation liquid composite bacterial agent for heavy metal contaminated soil according to claim 3, characterized in that, During the S1 stage, when fermentation reaches the middle of the logarithmic growth phase, add growth factors sodium citrate and sodium molybdate; after the S1 stage fermentation ends and the system pH stabilizes at 6.5±0.2, add growth factors glycerol and calcium phosphate; when the S3 stage is started, add growth factors potassium feldspar powder and humic acid.

5. The microbial ecological remediation liquid composite microbial agent for heavy metal contaminated soil according to claim 4, characterized in that, In S1, sodium citrate and sodium molybdate are added simultaneously and in batches using a pulsed slow-flow injection method. The concentration of sodium citrate is 0.5 g / L and the concentration of sodium molybdate is 10 μM. The sodium citrate is added in three batches, with each batch containing 1 / 3 of the total required amount, and the interval between batches is 2 hours. The glycerol concentration in S2 is 5 mM, and it is added continuously at a flow rate of 0.5 g / L·h to avoid local concentrations exceeding 10 mM; the calcium phosphate concentration is 2 g / L, added in 3 portions, each portion being 1 / 3 of the total required amount, with an interval of 4 hours. In S3, potassium feldspar powder and humic acid are added in two batches. The potassium feldspar powder has a particle size of 200 mesh and a concentration of 10 g / L. The humic acid is added after being dissolved in small molecular cluster water and has a concentration of 0.8 g / L. The two additions are 6 hours apart.

6. A micro-ecological remediation bio-fertilizer suitable for heavy metal contaminated soil, characterized in that, It is prepared using the micro-ecological remediation liquid compound microbial agent for heavy metal contaminated soil as described in any one of claims 1-5.

7. The microecological remediation bio-fertilizer for heavy metal contaminated soil according to claim 6, characterized in that, Preparation methods include: A1: The liquid compound microbial agent, electrochemical low-hertz water, and solid components (lignite powder, wheat bran, and soybean meal) described in claim 1 are added to a mixing system. After the mixture is evenly mixed, 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 through an intelligent material distribution system for a second aging and fermentation: the naturally piled material is deeply decomposed using the residual heat and residual microorganisms. After 10-12 days, the fermentation ends, and the micro-ecological remediation bio-fertilizer suitable for heavy metal contaminated soil is obtained.

8. The microecological remediation bio-fertilizer for heavy metal contaminated soil according to claim 7, characterized in that, In component A1, the total amount of solid components—lignite powder, wheat bran, and soybean meal—is 100%, with the following proportions: lignite powder 70-85%, wheat bran 8-15%, and soybean meal 8-20%. The lignite powder has a particle size of 80-100 mesh and an initial moisture content of 10-15%. The initial moisture content of the wheat bran and soybean meal is 8-12%. After the solid components are mixed evenly, electrolytic low-hertz water is added and stirred thoroughly. Simultaneously, a liquid compound microbial agent is added during the stirring process, at a rate of 0.8-10% of the total solid components, so that the total moisture content of the material reaches 50-60%.

9. The microecological remediation bio-fertilizer for heavy metal contaminated soil according to claim 8, characterized in that, The completion of the first fermentation stage A1 is indicated by the following: the temperature is maintained at the final stage temperature for 24 consecutive hours, the material moisture content is ≤40%, the material color is dark brown, there is no odor, it is loose when squeezed, and there is no stickiness.

10. The microecological remediation bio-fertilizer for heavy metal contaminated soil according to claim 9, characterized in that, The second fermentation in stage A2 is marked as follows: when 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 live bacteria count ≥200 million / g, the second fermentation is complete.

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