Universal soil micro-ecological restoration liquid complex microbial inoculant suitable for industrial production and biological nitrogen fertilizer of universal soil micro-ecological restoration liquid complex microbial inoculant

Abstract

The invention relates to the field of microbial agents, in particular to a universal soil micro-ecological restoration liquid composite microbial agent suitable for industrial production and a biological nitrogen fertilizer thereof. Comprising six functional strains: nitrogen-fixing bacteria, phosphate-solubilizing bacteria, potassium-solubilizing bacteria, growth-promoting bacteria, biocontrol bacteria and synergistic bacteria, the nitrogen-fixing bacteria are azotobacter chroococcum, the phosphate-solubilizing bacteria are pseudomonas fluorescens, the potassium-solubilizing bacteria are bacillus mucilaginosus, the growth-promoting bacteria are azotobacter braziliensis, the biocontrol bacteria are bacillus subtilis, and the synergistic bacteria are trichoderma asperellum; the liquid complex microbial inoculant is prepared from the six functional strains through liquid fermentation by using electrochemical low-Hertz water. The biological nitrogen fertilizer is prepared by using the liquid complex microbial inoculant, exerts the functions of nitrogen fixation, phosphate solubilization, potassium solubilization, growth promotion, biocontrol and synergy, also can reduce the dependence on chemical fertilizers, not only can improve the soil quality, but also achieves the synergy goals of crop yield increase, quality improvement and soil health.

Description

Technical Field

[0001] This invention relates to the field of microbial agents, specifically to a universal liquid compound microbial agent suitable for industrial production for soil microecological restoration and its bio-nitrogen fertilizer. Background Technology

[0002] In modern agricultural production, increased crop yields still largely rely on chemical fertilizers and pesticides. However, the utilization rate of chemical pesticides is low, with approximately 80% to 90% remaining in the environment. The excessive use of chemical fertilizers not only leads to soil compaction and increases planting costs but also causes serious environmental pollution. In recent years, using microbial inoculants to improve the soil micro-ecological environment and reduce reliance on chemical fertilizers has become a research and application hotspot. Soil properties vary greatly across different regions of my country. Currently, domestic soils are generally affected by various types of pollution, including soil compaction caused by chemical fertilizers, pesticide residues, and heavy metal pollution. Therefore, developing a microbial inoculant suitable for various soil types to achieve the synergistic goals of increased crop yields, improved quality, and improved soil health has become a current research focus. Summary of the Invention

[0003] To address the various environmental problems caused by excessive fertilizer application, and the fact that current single-strain or compound microbial agents are insufficient to fully meet the growth needs of various types of land and crops under the current widespread pollution conditions in China, as well as the problems of microbial imbalance, low yield, and difficulty in controlling product quality to optimize various conditions during the industrial preparation of compound microbial agents, this invention provides a universal liquid compound microbial agent for soil microecological restoration suitable for industrial production and its bio-nitrogen fertilizer, thus solving the above problems.

[0004] The technical solution of the present invention is as follows:

[0005] This invention provides a universal liquid compound microbial agent for soil microecological remediation suitable for industrial production, comprising the following six functional strains prepared by electrochemical low-Hertz water through three-stage sequential inoculation and fermentation: nitrogen-fixing bacteria: Azotobacter chroococcum, phosphate-solubilizing bacteria: Pseudomonas fluorescens, potassium-solubilizing bacteria: Paenibacillus mucilaginosus, growth-promoting bacteria: Azospirillumbrasilense, biocontrol bacteria: Bacillus subtilis, and synergistic bacteria: Trichoderma asperellum.

[0006] S1: Propagate nitrogen-fixing bacteria and growth-promoting bacteria to establish a nitrogen source base: Add electrolytic low-hertz water to the sterilized main fermenter and add pre-mixed seed liquid of *Azotobacter spp.* and *Azotobacter brasiliensis*. Adjust the fermentation conditions to the first-order fermentation conditions: pH = 7.0, dissolved oxygen 20-30%, temperature 30℃, fermentation time 22-24 hours.

[0007] 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 Pseudomonas fluorescens and Bacillus subtilis seed liquid to the main fermenter. The second sequential fermentation conditions are: pH=6.5, dissolved oxygen 30-40%, temperature 32℃, fermentation 16-18 hours;

[0008] 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 fermentation tank. After fermentation for 12 hours, add Trichoderma echinosporum seed liquid, turn on mechanical stirring and adjust to the third-sequence fermentation conditions: pH=5.5, dissolved oxygen 60%, temperature 28℃, fermentation for 34-36 hours.

[0009] When the number of viable bacteria in the main fermenter is ≥2×10 10 Fermentation ends when CFU / mL is reached.

[0010] Preferably, the mass ratio of *Azotobacter globosum*, *Pseudomonas fluorescens*, *Bacillus mucilaginosus*, *Azotobacter brasiliensis*, *Bacillus subtilis*, and *Trichoderma echinosporum* is 3:1:1:2:1:1.

[0011] Preferably, the seed culture preparation method is as follows: six 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 six primary seed tanks for independent culture.

[0012] 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 When CFU / mL, the seed cultures of *Azotobacter globosum* and *Azotobacter brasiliensis* were mixed at a volume ratio of 1:1 to obtain premixed seed culture 1. The seed cultures of *Pseudomonas fluorescens* and *Bacillus subtilis* were mixed at a volume ratio of 1:1 to obtain premixed seed culture 2. After incubation at 30°C for 30 minutes, the two premixed cultures were used for sequential fermentation.

[0013] 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;

[0014] The nitrogen-fixing bacteria and growth-promoting bacteria were prepared using Ashube medium, the phosphate-solubilizing bacteria were prepared using KB medium, the biocontrol bacteria were prepared using LB medium, the potassium-solubilizing bacteria were prepared using potassium-solubilizing medium, and Trichoderma acicularis was prepared using PDA medium; all five media were prepared using electrochemical low-hertz water.

[0015] The inoculation amounts of the premixed seed liquid 1, premixed seed liquid 2, Bacillus mucilaginosus seed liquid, and Trichoderma echinosporum seed liquid are 9.8%-10.2%, 4.8%-5.2%, 4.8%-5.2%, and 2.8%-3.2% of the fermentation volume, respectively. The inoculation amount of electrochemical low-hertz water is 76.4%-77.6% of the fermentation volume. The electrochemical low-hertz water has a molecular cluster of ≤6 molecules and a pH of 7.0–7.5.

[0016] Preferably, during the S1 stage, when fermentation reaches the middle of the logarithmic growth phase, growth factors succinic acid and sodium molybdate are added; after the S2 stage fermentation is completed and the system pH is stable, growth factors glycerol and calcium phosphate are added; when the S3 stage is started, growth factor potassium feldspar powder is added.

[0017] Preferably, succinic acid and sodium molybdate in S1 are added simultaneously and in batches using a pulsed slow-flow injection method. The concentration of succinic acid is 0.01%, and the concentration of sodium molybdate is 10 μM. The addition is done in three batches, with each batch containing 1 / 3 of the total required amount, and the interval between batches is 2 hours.

[0018] In S2, 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; 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 between each batch.

[0019] The potassium feldspar powder in S3 is added in two batches. The potassium feldspar powder has a particle size of 200 mesh and a concentration of 10 g / L. The two additions are 6 hours apart.

[0020] This invention also provides a universal soil microecological restoration bio-nitrogen fertilizer suitable for industrial production, characterized in that it is prepared using the above-mentioned universal soil microecological restoration liquid compound microbial agent suitable for industrial production.

[0021] Preferably, the preparation method includes:

[0022] A1: The liquid compound microbial agent, electrochemical low-hertz water, and solid components (lignite powder, wheat bran, and soybean meal) are added to the mixing system. After uniform mixing, the mixture is conveyed to the solid-state fermentation system production line via 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.

[0023] 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 allowed to undergo deep decomposition using residual heat and residual microorganisms. Fermentation continues for 10-12 days until the end of the fermentation, thus obtaining the universal soil micro-ecological restoration bio-nitrogen fertilizer. The sign of the end of fermentation is: when the material naturally cools down to a moisture content of 15-29%, has a uniform dark brown color, loose texture, a muddy 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.

[0024] 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%.

[0025] 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.

[0026] Preferably, the completion of the second fermentation in stage A2 is indicated by the following: when the material naturally cools to a moisture content of 15-29%, has a uniform dark brown color, a loose texture, an earthy smell without other off-odors, a pH of 6.5-7.5, and a beneficial viable bacteria count ≥2×10⁻⁶. 10 When the concentration reaches / g, the second fermentation ends.

[0027] The beneficial effects of this invention are as follows:

[0028] This invention provides a universal liquid compound microbial agent for soil microecological remediation suitable for industrial production. Through a design of grouping and sequential fermentation in the main tank, it organically combines strains with different fermentation conditions that exhibit mutual growth within the same timeframe and metabolic complementarity between different timeframes. This allows the liquid compound microbial agent to comprehensively meet the growth needs of various types of land and crops under the current widespread pollution conditions in China. The liquid compound microbial agent achieves a synergistic effect of soil remediation and nutrient enhancement through the scientific combination of six functional bacteria. Firstly, it utilizes a synergistic activation mechanism of nutrient elements. Nitrogen-fixing bacteria (Brachystomum globosum) convert atmospheric nitrogen into ammonium nitrogen, providing a nitrogen source for the proliferation of other microbial communities, especially significantly enhancing the effect on legumes. Phosphate-solubilizing bacteria (Pseudomonas fluorescens) secrete organic acids to dissolve fixed phosphates in the soil, releasing soluble phosphorus. This, combined with potassium-solubilizing bacteria (Bacillus mucilaginosus), enhances the release efficiency of phosphorus and potassium. Bacillus mucilaginosus decomposes silicate minerals to release potassium ions, and its secreted extracellular polysaccharides can also enhance soil aggregate structure and alleviate compaction. Secondly, there is a synergistic mechanism of stress resistance, repair, and growth promotion. The growth-promoting bacterium *Azotobacter brasiliensis* secretes auxins (such as IAA) to stimulate root development, expand nutrient absorption area, and form a closed-loop nitrogen supply system with the nitrogen-fixing bacteria. The biocontrol bacterium *Bacillus subtilis* produces lipopeptide antimicrobial substances (such as surfactants) to inhibit soil-borne pathogens such as *Fusarium*, while simultaneously activating plant systemic resistance. The synergistic bacterium *Trichoderma echinococcus* degrades the cell walls of pathogenic fungi through hyperparasitism, and its secreted chitinase can decompose pesticide residue molecular chains. Combined use with *Bacillus subtilis* can improve disease control efficiency. Thirdly, there is a microecological cycle mechanism: *Azotobacter chrysogenum* provides a nitrogen source, while *Pseudomonas fluorescens* and *Bacillus mucilaginosus* release mineral nutrients, forming a self-sufficient microecological cycle and achieving nutrient complementarity. *Trichoderma echinococcus* hyphae provide colonization channels for other fungal communities, and *Bacillus subtilis* forms a bioprotective film in the plant rhizosphere, providing space for rapid proliferation of the fungal species.

[0029] For compacted soil, *Bacillus mucilaginosus* secretes extracellular polysaccharides to cement soil particles, which, together with the hyphal network of *Trichoderma echinosporum*, weave a biological framework, promoting the formation of microaggregates and increasing soil porosity. Humic acid produced by *Azotobacter chrysogenum* enhances aggregate stability and prevents soil compaction from recurring. *Pseudomonas fluorescens* secretes citric acid to dissolve carbonate crystals in the compacted layer, while *Bacillus mucilaginosus* releases silicate enzymes to decompose mineral clay particles, breaking down the hardened barrier. For soil contaminated with heavy metals, the liquid composite bacterial agent remediates heavy metal-contaminated soil through two pathways: bio-passivation and transformation, and stable storage mechanisms. *Pseudomonas fluorescens* uses redox reactions to convert highly toxic Cr... 6+ Transformed into low-migration Cr 3+ Bacillus subtilis chelates Cd through the carboxyl groups of its cell wall. 2+Heavy metal ions are reduced, thus lowering their bioavailability. *Azotobacter chrysogenum* secretes humic acid to form stable complexes with heavy metals, while *Trichoderma echinococcus* accumulates pollutant ions through hyphae, synergistically reducing soil heavy metal activity. For soils with pesticide residues, *Trichoderma echinococcus* secretes chitinase and protease to cleave the phosphate ester bonds of organophosphorus pesticides, while *Bacillus subtilis* produces lipopeptide surfactants to emulsify pesticide molecules, improving their degradation efficiency. *Pseudomonas fluorescens* and *Azotobacter brasiliensis* jointly mineralize the carbon skeleton of pesticides, converting them into CO2 and water, shortening the degradation cycle of residual toxins.

[0030] Regarding the stability of the microbial agent's efficacy, the extracellular polysaccharides secreted by *Bacillus mucilaginosus* in the agent combine with *Trichoderma echinococcosis* hyphae to form a biogel layer that encapsulates the bacterial community, buffering environmental stresses (such as pH fluctuations and heavy metal shocks) and ensuring the survival rate of the bacterial community in contaminated soil. *Bacillus subtilis* produces lipopeptide antimicrobial substances to inhibit competing bacteria, enhancing the community's adaptability to the environment. *Pseudomonas fluorescens* tolerates heavy metal ions (such as Cd) through its oxidase system. 2+ This ensures the colonization advantage of the core microbial community. Combined with a self-sufficient microecological cycle, the stability of this microbial agent's effectiveness far surpasses that of ordinary microbial agents.

[0031] Regarding the application rate of the microbial agent, the IAA hormone secreted by *Azotobacter brasiliensis* expands the root surface area of ​​crops, while the extracellular polysaccharides of *Bacillus mucilaginosus* enhance the adsorption capacity of rhizosphere nutrients, thus jointly improving the colonization efficiency of the microbial agent. The mycelial network of *Trichoderma echinosporum* carries nitrogen-fixing spores to penetrate soil fissures, and the biofilm of *Bacillus subtilis* encapsulates phosphate-solubilizing bacteria to prevent inactivation, increasing the repair area per unit of inoculum. *Pseudomonas fluorescens* activates plant systemic resistance, forming a dual "physical and biochemical" protection with the antimicrobial peptides of *Bacillus subtilis* to resist microbial imbalance caused by temperature and humidity fluctuations. The secretions of *Bacillus mucilaginosus* combine with the metabolic colloids of *Trichoderma echinosporum* to form slow-release microcapsules, continuously releasing active bacteria in the soil. The synergistic effect of these multiple functions allows the application rate of the microbial agent provided by this invention to be far lower than that of conventional microbial agents.

[0032] From the perspective of the formulation of liquid compound microbial agents, nitrogen-fixing bacteria (Azotobacter chrysogenum) account for the highest proportion. As the core of nitrogen supply, its efficient nitrogen-fixing ability provides the basic nitrogen source for the entire microbial community. Growth-promoting bacteria (Azospirillum brasiliensis) has both nitrogen-fixing and indoleacetic acid (IAA) synthesis functions, accounting for 2 parts, which can enhance the crop growth-promoting effect. Phosphate-solubilizing bacteria (Pseudomonas fluorescens) and potassium-solubilizing bacteria (Bacillus mucilaginosus) each account for 1 part, which meets the soil's phosphorus and potassium element activation needs. Biocontrol bacteria (Bacillus subtilis) and synergistic bacteria (Trichoderma echinosporum) each account for 1 part. Bacillus subtilis inhibits pathogens by secreting antimicrobial peptides, while Trichoderma echinosporum can promote the stability of the microbial agent and the decomposition of organic matter. The proportion of the two is lower than that of bacteria to avoid interspecific competition and inhibition. This invention forms a complete microbial ecosystem through the compatibility and synergistic fermentation of various functional strains, including nitrogen fixation, phosphorus solubilization, potassium solubilization, growth promotion, biocontrol, and synergistic effects. This improves soil nutrient utilization, enhances crop disease resistance and yield, and is widely applicable to various types of land and crop growth needs under conditions of widespread pollution. In particular, the application of electrochemical low-hertz water throughout the entire process ensures a stable increase in the yield of the microbial agent, which is significantly higher than that of single-strain culture in separate tanks. Combined with subsequent industrial automated control of microbial fertilizer production, the microbial fertilizer has strong universality and stable efficacy.

[0033] By designing fermentation conditions for each time sequence, the problems of microbial imbalance, low yield, and difficulty in controlling product quality that easily occur in the industrial preparation of liquid compound microbial agents were solved, thus achieving optimization of various conditions.

[0034] This invention groups six functional bacteria according to their growth requirements and performs time-sequential culture:

[0035] The first inoculation sequence consists of nitrogen-fixing bacteria—*Azotobacter chrysogenum* and *Azospirillum brasiliensis*. Both are aerobic nitrogen-fixing bacteria, and their nitrogenase, produced by metabolism, is sensitive to oxygen, requiring a microaerobic environment. Their optimal pH is neutral, and they grow relatively slowly. The first-sequence fermentation conditions set in this invention are: dissolved oxygen 20-30%, which meets the basic aerobic requirements of both bacteria, while avoiding excessive dissolved oxygen levels that inhibit nitrogenase activity; pH ​​set at 7.0 to match their preference for a neutral environment; and long-term fermentation at 30°C to accommodate their slow growth characteristics. Nitrogen-fixing bacteria fermentation requires a low-nitrogen environment, while a high-nitrogen environment will inhibit their growth. At this time, the fermenter only contains electrolytic low-hertz water, without any culture medium, and therefore no other nitrogen source input. Thus, placing nitrogen-fixing bacteria in the first-sequence fermentation can increase the reproduction rate and provide the nitrogen source basis required for subsequent bacterial fermentation. The humidity of the top space of the fermenter is set to 92%±1%, which effectively maintains the high water activity required for nitrogenase synthesis by Azotobacter chrysogenum and cell metabolism by Azotobacter brasiliensis during the secretion of plant growth hormones, and prevents nitrogen fixation efficiency from decreasing or the synthesis of growth-promoting substances from being hindered due to water evaporation stress.

[0036] The second-order inoculation involves phosphate-solubilizing bacteria—*Pseudomonas fluorescens*, and biocontrol bacteria—*Bacillus subtilis*. Both are 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 order as follows: dissolved oxygen 30-40% to support the high aerobic 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. Furthermore, both fermentations rely on the nitrogen source produced in the first-order fermentation to synthesize metabolic enzymes, thus efficiently utilizing the nitrogen source from the early stage. The activated phosphorus produced during fermentation provides nutrients for the third-order strains; if this stage is advanced, phosphorus utilization will significantly decrease. The humidity in the top space of the second-order fermenter is maintained at 92%±1%, precisely matching the physiological needs of phosphate-solubilizing bacteria *Pseudomonas fluorescens* during phosphatase secretion and biocontrol bacteria *Bacillus subtilis* during the large-scale synthesis of antimicrobial peptides, avoiding the decline in extracellular enzyme activity or membrane transport efficiency caused by humidity fluctuations.

[0037] In the third time step, *Bacillus mucilaginosus* seed culture is added to the main fermenter, and after 12 hours of fermentation, *Trichoderma echinococcus* seed culture is inoculated. *Bacillus mucilaginosus*, as a typical phosphate-solubilizing and potassium-solubilizing bacterium, possesses acid-resistant metabolic characteristics; *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 delayed 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 proliferation rhythm of *Bacillus mucilaginosus*, maintaining the ecological balance of the bacterial and fungal populations in the fermentation system, laying the foundation for synergistic function between the two. In this stage, fermentation parameters are precisely set according to synergistic metabolic requirements: dissolved oxygen concentration is controlled at 60% to fully meet the strict aerobic metabolic needs of *Trichoderma echinococcosis*; pH is adjusted to 5.5, which is both suitable for the optimal acidic environment for fungal enzyme production and compatible with the acid-resistant characteristics of *Bacillus mucilaginosus*; fermentation temperature is maintained at 28℃, a temperature range that simultaneously meets the optimal growth requirements of both fungi and bacteria, ensuring the stability of functional metabolism during long-term fermentation. The humidity in the top space of the third-order fermenter is controlled at 88%±1%. While enhancing oxygen supply efficiency through mechanical stirring, this humidity environment precisely matches the metabolic characteristics of *Bacillus mucilaginosus* (a potassium-solubilizing strain) in secreting extracellular polysaccharides to decompose silicates, as well as the life cycle requirements of *Trichoderma echinococcosis* in forming conidia and synthesizing cellulase. This promotes mineral activation efficiency and the construction of a synergistic hyphal network, avoiding delayed spore formation or dilution of metabolic products due to excessive humidity, thus ensuring the functional stability and application effect of the liquid compound microbial agent.

[0038] In particular, this invention achieves low-oxygen protection for nitrogen-fixing bacteria and high-oxygen protection for Trichoderma by setting grouping and three time-series differentiated fermentation conditions; 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 the requirements of phosphate-solubilizing bacteria for an acidic environment.

[0039] This invention uses the same independent main fermenter, with each group undergoing different sequential fermentation in parallel, avoiding idle tanks and reducing the probability of cross-contamination and production costs.

[0040] 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, electrochemical 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-nitrogen ratio, thus establishing a nitrogen source basis; it also relieves phosphorus limitation by promoting the secretion of organic acids (such as gluconic acid and citric acid) and phytase by *Pseudomonas fluorescens*, thereby dissolving insoluble phosphorus (such as calcium phosphate and apatite); it releases mineral potassium and intensifies mineral decomposition by enhancing the adsorption and acidolysis capacity of *Bacillus mucilaginosus* on silicate minerals; and it promotes plant growth by promoting 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.

[0041] The three groupings and corresponding time sequences designed in the liquid compound microbial agent 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 earlier propagating strains do not adversely affect the later propagating strains, allowing them to be efficiently utilized by the later propagating strains to promote growth. Furthermore, due to the rational design, the secondary propagation process of the liquid compound microbial agent (i.e., the time-sequential 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, thus simplifying the production process and effectively reducing production costs.

[0042] In the preparation of the liquid compound bacterial agent, succinic acid and sodium molybdate are added to the system during the first sequential fermentation at the mid-logarithmic growth phase (12-16 hours). At this stage, the bacterial metabolic activity is highest, and the absorption efficiency of nutrients is also at its peak. Succinic acid, as an intermediate in the tricarboxylic acid cycle (TCA), directly provides energy and a carbon skeleton for the bacteria; sodium molybdate, as a core cofactor of nitrogenase, avoids metal toxicity at low concentrations and maintains consistency with the concentration in the basal culture medium of nitrogen-fixing bacteria, thus helping to maintain nitrogenase stability. Therefore, a pulsed addition method is used to avoid excessively high instantaneous concentrations that could inhibit bacterial growth.

[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 prevent local concentrations exceeding 10 mM from inhibiting cell growth, glycerol is added continuously (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, avoiding precipitation and uneven dissolved oxygen distribution caused by a single high-concentration addition. Furthermore, glycerol and calcium phosphate have a synergistic effect: glycerol provides energy, and calcium phosphate serves as a substrate; simultaneous addition of both synergistically activates the phosphate-solubilizing metabolic pathway.

[0044] At the start of the third fermentation, add 200-mesh potassium feldspar powder (10 g / L) in two separate additions (6 hours apart) to avoid excessive addition leading to increased system viscosity and reduced dissolved oxygen efficiency. The oxalic acid secreted by *Trichoderma* can synergistically decompose the silicate structure in the potassium feldspar powder. Once the *Trichoderma echinosporum* 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 sufficient contact between the potassium feldspar powder and the fungal cells, thus improving the fungal reproduction efficiency.

[0045] This invention provides a universal soil micro-ecological restoration bio-nitrogen fertilizer suitable for industrial production. The raw materials are lignite powder, wheat bran, soybean meal, liquid compound microbial agent, and electrochemical low-Hertz water. While meeting the requirements of organic matter >40%, live bacteria >200 million / gram, and fermentation raw material moisture content between 50% and 60%, the proportion of lignite powder is maximized to 85% by reducing wheat bran and soybean meal to the minimum necessary amount (ensuring microbial nutrition), significantly reducing raw material costs. It is prepared using a secondary fermentation process, incorporating micro-aeration technology to replace traditional turning during production, reducing production costs and energy consumption. Combined with an intelligent material distribution system, it achieves continuous production without human intervention, suitable for large-scale production lines. Furthermore, intermittent high-oxygen stimulation significantly improves the efficiency of solid-state fermentation and product quality. Because it is prepared using the aforementioned liquid compound microbial agent, this bio-nitrogen fertilizer possesses multiple functions such as nitrogen fixation, phosphorus solubilization, potassium solubilization, growth promotion, biocontrol, and synergistic effects. It can form a complete microbial ecosystem in the soil, effectively alleviating soil compaction and creating a soil environment rich in microorganisms and various nutrients. This enhances crop disease resistance and yield, and it is widely applicable to various types of land and crop growth needs under conditions of widespread pollution. It not only improves soil quality but also achieves the synergistic goals of increased crop yield, improved quality, and soil health. Furthermore, the preparation process of the universal soil microecological restoration bio-nitrogen fertilizer provided by this invention is a comprehensive design aimed at industrial production. Therefore, in actual crop production, the application amount of this microbial fertilizer can be reduced by 65.14%-98.14% compared to urea fertilizer for the same area, while increasing the available phosphorus content in the soil by 41.67% and reducing the number of pathogenic bacteria by 50%. This solves the problem of unstable efficacy and large fertilizer application rates in bio-agents and fertilizers made from single strains or simple combinations of strains in practical applications. 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: A universally applicable liquid compound microbial agent for soil microecological restoration suitable for industrial production

[0048] The universal soil microecological remediation liquid compound bacterial agent suitable for industrial production described in this embodiment includes six functional bacterial strains: nitrogen-fixing bacteria, phosphate-solubilizing bacteria, potassium-solubilizing bacteria, growth-promoting bacteria, biocontrol bacteria, and synergistic bacteria. The nitrogen-fixing bacteria is *Azotobacter chroococcum*, the phosphate-solubilizing bacteria is *Pseudomonas fluorescens*, the potassium-solubilizing bacteria is *Paenibacillus mucilaginosus*, the growth-promoting bacteria is *Azospirillum brasilense*, the biocontrol bacteria is *Bacillus subtilis*, and the synergistic bacteria is *Trichoderma asperellum*. The total effective bacterial count is ≥2 × 10⁻⁶. 10 CFU / mL), fungal spore count ≥1×10⁻⁶ 8 Spores / mL; the mass ratio of the aforementioned *Azotobacter globosum*, *Pseudomonas fluorescens*, *Bacillus mucilaginosus*, *Azotobacter brasiliensis*, *Bacillus subtilis*, and *Trichoderma echinosporum* is 3:1:1:2:1:1.

[0049] The liquid compound microbial agent is prepared by liquid fermentation of the six functional strains using electrolytic low-hertz water. The liquid fermentation specifically includes the following stages:

[0050] Six bacterial strains were inoculated from preservation slant agar to specific agar slant 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, and *Trichoderma echinosporum* on PDA medium. All five media were prepared using electrochemically controlled 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 six primary seed tanks for culture.

[0051] When the bacterial count is ≥5×10 8 At a CFU / mL concentration, premixed seed solutions 1 were prepared by mixing *Azotobacter chamaescoparia* and *Azotobacter brasiliensis* seed solutions at a 1:1 volume ratio, and premixed seed solutions 2 were prepared by mixing *Pseudomonas fluorescens* and *Bacillus subtilis* seed solutions at a 1:1 volume ratio. 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 satisfactory. 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.

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

[0053] S1: Propagation of nitrogen-fixing bacteria to establish a nitrogen source: Add electrolytic low-hertz water to the sterilized main fermenter, and add pre-mixed seed culture of *Azotobacter blazei* and *Azotobacter brasiliensis*, with an inoculation volume of 9.8%-10.2% 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 22-24 hours; when fermentation reaches the middle of the logarithmic growth phase, add the growth factors succinic acid and sodium molybdate. Succinic acid and sodium molybdate are added simultaneously using a pulsed slow-flow injection method, with the concentration of succinic acid being 0.01% and the concentration of sodium molybdate being 10μM. Each addition is 1 / 3 of the total required amount, with an interval of 2 hours, for a total of 3 times.

[0054] S2: Introducing phosphate-solubilizing bacteria and biocontrol bacteria to alleviate phosphorus limitation: After the S1 stage, adjust to the second sequential fermentation conditions, and add a pre-mixed seed culture of *Pseudomonas fluorescens* and *Bacillus subtilis* to the main fermenter at an inoculation volume of 4.8%-5.2% of the fermentation volume. The second sequential fermentation conditions are: pH 6.5, dissolved oxygen 30-40%, temperature 32℃, humidity at the top of the fermenter 92%±1%, and fermentation time 16-18 hours. After fermentation is complete and the system pH is stable, add the growth factors glycerol and calcium phosphate. 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 between each batch.

[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 4.8%-5.2% of the fermentation volume; after 12 hours of fermentation, Trichoderma echinosporum seed liquid is added at an inoculation rate of 2.8%-3.2% 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℃, humidity at the top of the fermenter 88%±1%, fermentation time 34-36 hours; after fermentation is complete and the system pH is stable, growth factors glycerol and calcium phosphate are added. Potassium feldspar powder with a particle size of 200 mesh and a concentration of 10 g / L is added 6 hours apart. When the viable count in the main fermenter is ≥2×10⁻⁶... 10 Fermentation ends when CFU / mL, yielding the liquid compound microbial agent.

[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: Universal Soil Microecological Restoration Bio-nitrogen Fertilizer Suitable for Industrial Production

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

[0062] In a specific embodiment of the present invention, the raw materials of the bio-nitrogen 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 particle size of the lignite powder is 80-100 mesh, and the initial moisture content is 10-15%; the initial moisture content of the wheat bran and soybean meal is 8-12% each. After the above solid components are mixed evenly, electrochemical low-hertz water is added and stirred thoroughly. At the same time, the liquid compound microbial agent is added simultaneously during the stirring process, and the amount added is calculated as 0.8-10% of the total amount of solid components, so that the total moisture content of the material reaches 50-60%.

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

[0064] A1: The liquid compound microbial agent, electrochemical low-hertz water, and solid components (lignite powder, wheat bran, and soybean meal) 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 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; and the final stage temperature is 40-45℃, maintained for 22-24 hours. When the temperature remains constant 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, the fermentation of stage A1 is complete.

[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 universal soil micro-ecological restoration biological nitrogen fertilizer 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 bacterial agent prepared according to the method in 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 43.8% compared to the control group; at the end of the second time step, the number increased by 38.5%; and at the end of the third time step, the number increased by 31.4%. This indicates that electrochemical low-hertz water can effectively improve fermentation efficiency.

[0070] Table 1. Viable bacterial count

[0071]

[0072] Experiment Example 2: Effects of using bio-nitrogen fertilizer for soil microecological restoration

[0073] 2.1 Maize Experiment

[0074] (1) Test location:

[0075] The soil in Taocheng District, Hengshui City, Hebei Province is classified as alluvial soil (FAO classification). The pH value is 7.2±0.3 (determined by potentiometric method). The nutrient indicators are available nitrogen 78.2±3.1 mg / kg (Kjeldahl method) and available phosphorus 12.3±0.8 mg / kg (Olsen method).

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

[0077] Processing Group 1:

[0078] Fertilizer: The amount of liquid compound microbial agent added to the bio-nitrogen fertilizer 1 prepared according to the preparation method of Example 2 is 0.8%.

[0079] Application method: Before planting, use Stanley commercial organic fertilizer (organic matter ≥30%) as base fertilizer (300 kg / mu). Before planting, use biological nitrogen fertilizer 1 to mix the seeds, with a dosage of 0.9 kg / mu. From the jointing stage to the large trumpet stage, spray the supernatant obtained by dissolving biological nitrogen fertilizer 1 in tap water onto each group of fertilizers, with a dosage of 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 biological nitrogen 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 Corn Special Compound Fertilizer (22-18-10), Stanley Urea (≥46%), Stanley Commercial Organic Fertilizer (Organic Matter ≥30%).

[0088] Application method: Use 300 kg / mu Stanley organic fertilizer and 50 kg / mu Zhonghua chemical fertilizer corn-specific compound fertilizer as base fertilizer; apply Stanley urea top dressing at the large trumpet stage, 30 kg / mu; apply Stanley organic fertilizer top dressing at the tasseling and silking stage, 10 kg / mu.

[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. The variety planted was "Zhengdan 958", and the field management of each group was consistent.

[0091] (4) Experimental results:

[0092] The experimental results are shown in Table 2 below: Compared with treatment group 2, treatment group 1 showed an increase in plant height of 4.07%, leaf area index of 4.00%, shortened tasseling and silking time of 3.5%, increased number of aerial roots of 18.18%, increased stem diameter of 6.10%, increased yield per unit area of ​​3.79%, increased available phosphorus content in the soil of 11.76%, and reduced the incidence of stem rot by 37.5%. The bio-nitrogen fertilizer provided by this invention effectively promotes the growth and yield of maize and significantly reduces the incidence of maize stem rot. Under the same application method and dosage, the bio-nitrogen fertilizer 1 in treatment group 1 is significantly more effective than bio-nitrogen fertilizer 2 in treatment group 2, nitrogen-fixing fertilizer in control group 1, and commercial organic fertilizer in control group 2.

[0093] Table 2 Results of the maize planting experiment

[0094]

[0095] 2.2 Wheat Experiment

[0096] (1) Test location:

[0097] Tanghe County, Nanyang City, Henan Province: Soil properties: yellow-brown soil, pH: 6.8-7.3, organic matter content: 1.2%-1.5%, available nitrogen content: 70-85 mg / kg, available phosphorus content: 15-20 mg / kg, available potassium content: 110-130 mg / kg.

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

[0099] Processing Group 1:

[0100] Fertilizer: The biological nitrogen 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: Before sowing, use 400 kg / mu of Nanyang Nongfeng commercial organic fertilizer as base fertilizer, and use biological nitrogen fertilizer 1 for seed dressing at a rate of 1.6 kg / mu. During the greening period, apply biological nitrogen fertilizer 1 at a rate of 8 kg / mu. During the jointing period, dissolve biological nitrogen fertilizer 1 in water, spray and take the supernatant at a rate of 10 kg / mu.

[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 biological nitrogen 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 Compound Fertilizer (15-15-15), Sinochem Fertilizer Urea (≥46.4%), Nanyang Nongfeng Commercial Organic Fertilizer (Organic Matter ≥35%).

[0110] Application method: Use 400 kg / mu of Nanyang Nongfeng commercial organic fertilizer and 50 kg / mu of Sinochem compound fertilizer (15-15-15) as base fertilizer; use 20 kg / mu of Sinochem urea as top dressing during the greening and jointing stage.

[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 variety planted was "Aikang 58" (conventional thousand-grain weight 40-42g), and the field management of each group was the same.

[0113] (4) Test results

[0114] The experimental results are shown in Table 3 below: Compared with treatment group 2, treatment group 1 showed an increase of 9.37% in effective tiller number, 18.75% in grain filling rate, 5.81% in thousand-grain weight, 5.54% in yield per unit area, a 33.3% decrease in sheath blight incidence, and a 40.0% decrease in Fusarium head blight incidence. The bio-nitrogen fertilizer provided by this invention increases the number of effective tillers in wheat, improves grain filling rate, thousand-grain weight, and yield per unit area, and reduces the incidence of sheath blight and Fusarium head blight. Under the same application method and dosage, the growth-promoting effect of bio-nitrogen fertilizer 1 in treatment group 1 was significantly better than that of bio-nitrogen fertilizer 2 in treatment group 2, nitrogen-fixing fertilizer in control group 1, and commercial organic fertilizer in control group 2.

[0115] Table 3 Wheat Experiment Results

[0116]

[0117] 2.3 Cucumber Experiment

[0118] (1) Test location:

[0119] Linyi City, Shandong Province: Soil properties: sandy loam, pH 6.5-7.0, organic matter 1.0%-1.3%, EC value 0.8-1.2 mS / cm.

[0120] (2) Test fertilizers and fertilization methods:

[0121] Processing Group 1:

[0122] Fertilizer: The biological nitrogen fertilizer 1 prepared according to the preparation method of Example 2 has a liquid compound microbial agent added at a rate of 5%.

[0123] Application method: Before planting, use 1000 kg / mu of Linyi Keda organic fertilizer and 40 kg / mu of Sinochem compound fertilizer as base fertilizer; when transplanting, apply bio-nitrogen fertilizer 1 in furrows at a rate of 60 kg / mu. During the initial flowering period, early fruiting period, and peak fruiting period, apply top dressing by drip irrigation (dissolve bio-nitrogen fertilizer 1 in water and apply the supernatant by drip irrigation) at a rate of 8 kg / mu.

[0124] Processing Group 2:

[0125] 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 biological nitrogen fertilizer 2.

[0126] Application method: Same as treatment group 1.

[0127] Control group 1:

[0128] 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.

[0129] Application method: Same as treatment group 1.

[0130] Control group 2:

[0131] Fertilizers: Sinochem Fertilizer Compound Fertilizer (17-17-17), Sinochem Fertilizer Urea (≥46.4%), Linyi Keda Commercial Organic Fertilizer (Organic Matter ≥45%).

[0132] Application method: Use 1000 kg / mu of Linyi Keda organic fertilizer and 40 kg / mu of Sinochem compound fertilizer as base fertilizer; from seedling stage to early flowering stage, use 10 kg / mu of Sinochem compound fertilizer (17-17-17) for drip irrigation; during the peak fruiting period, use 10 kg / mu of Sinochem urea and 15 kg / mu of Sinochem compound fertilizer (17-17-17) for top dressing.

[0133] (3) Experimental design:

[0134] Greenhouse cultivation was carried out, with each group covering 4 mu (approximately 0.67 hectares) and repeated 3 times. The variety planted was "Jinchun No. 4," and field management was consistent across all groups.

[0135] (4) Test results

[0136] The experimental results are shown in Table 4 below: Compared with the control group, the number of fruits per plant increased by 18.18% in the treatment group.

[0137] The commercial fruit rate increased by 6.67%, yield per unit area increased by 7.14%, nitrate content in fruits decreased by 11.1%, vitamin C content in fruits increased by 11.60%, and the incidence of Fusarium oxysporum decreased by 80.0%. The soil microecological restoration bio-nitrogen fertilizer provided by this invention effectively promotes the growth, yield, and quality of cucumbers, and reduces the number of Fusarium oxysporum in the soil and the nitrate content in the fruits. Under the same application method and dosage, the bio-nitrogen fertilizer 1 in treatment group 1 was significantly more effective than bio-nitrogen fertilizer 2 in treatment group 2, nitrogen-fixing fertilizer in control group 1, and commercial organic fertilizer in control group 2.

[0138] Table 4 Results of cucumber cultivation experiment

[0139]

[0140] 3. Summary

[0141] 3.1 Comparison of the effects of bio-nitrogen fertilizer prepared using liquid compound microbial agents and single nitrogen-fixing microbial agents

[0142] This invention uses a combination of six functional strains—Azotobacter chrysogenum (nitrogen fixation), Pseudomonas fluorescens (phosphate solubilization), Bacillus mucilaginosus (potassium solubilization), Azotobacter brasiliensis (growth promotion), Bacillus subtilis (biocontrol), and Trichoderma echinosporum (antagonism)—to prepare a bio-nitrogen fertilizer (treatment group 1). Through the synergistic effect of "nitrogen fixation-phosphate and potassium solubilization-growth promotion-biocontrol," the growth promotion effect is significantly better than that of a single nitrogen-fixing bacterium inoculant (control group 1).

[0143] In the maize experiment: compared with control group 1, treatment group 1 showed an 8.29% increase in yield per unit area, a 35.71% increase in available phosphorus in the soil, and a 58.3% decrease in the incidence of stalk rot (Table 2). Mechanism of action analysis: *Azotobacter globosum* and *Azotobacter brasiliensis* provide the nitrogen source, exhibiting higher nitrogen fixation efficiency than single azotobacter species; *Pseudomonas fluorescens* and *Bacillus mucilaginosus* release phosphorus and potassium from the soil; and *Bacillus subtilis* and *Trichoderma echinosporum* inhibit stalk rot pathogens.

[0144] In wheat trials: Compared to control group 1, treatment group 1 showed a 16.67% increase in effective tiller number and a 50% decrease in sheath blight index (Table 3). Mechanism of action analysis: *Azotobacter brasiliensis* and *Bacillus subtilis* promoted tillering, *Pseudomonas fluorescens* and *Bacillus mucilaginosus* increased grain-filling rate, and *Trichoderma echinosporum* antagonized the sheath blight pathogen.

[0145] In the cucumber experiment: compared with control group 1, treatment group 1 showed a 44.44% increase in the number of fruits per plant, a 90% decrease in the number of Fusarium oxysporum in the soil, and a 24.69% increase in vitamin C in the fruits (Table 4). Mechanism of action analysis: Bacillus subtilis and Trichoderma echinosporum inhibited the growth of Fusarium oxysporum, Pseudomonas fluorescens and Bacillus mucilaginosus promoted cucumber fruit setting, and Azotobacter chrysogenum and Azospirillum brasiliensis improved fruit quality.

[0146] 3.2 Comparison of the effects of electrochemical low-hertz water and ordinary sterilized water

[0147] This invention uses electrochemical low-hertz water with a half-width at half-maximum (WHM) of ≤100Hz to prepare bio-nitrogen 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-nitrogen fertilizer 2 (treatment group 2) made with ordinary water.

[0148] In the maize experiment, compared with treatment group 2, treatment group 1 showed a 4.07% increase in plant height and a 3.79% increase in yield per unit area (Table 2). Mechanism of action analysis: Electrochemically treated low-Hertz water improved the proliferation efficiency of live bacteria in the inoculant, enhancing nutrient absorption by maize roots.

[0149] In the wheat experiment, compared with treatment group 2, treatment group 1 showed a 5.81% increase in thousand-grain weight and an 18.75% increase in grain filling rate (Table 3). Mechanism of action: Electrochemical low-Hertz water enhances the adhesion of the bacterial agent to wheat roots, resulting in a higher number of viable bacteria in the roots after application compared to ordinary water, thus promoting the absorption of nitrogen, phosphorus, and potassium by the crop.

[0150] In cucumber experiments, compared with treatment group 2, treatment group 1 showed an 11.60% increase in vitamin C content and an 11.1% decrease in nitrate content in the fruit (Table 4). Mechanism of action: Electrochemical low-Hertz water improves the diffusion of the inoculant in the soil, resulting in a higher number of viable bacteria in the rhizosphere of crops after drip irrigation compared to ordinary water, thus reducing nitrate accumulation.

[0151] 3.3 Comparison of bio-nitrogen fertilizer with conventional organic fertilizer and chemical fertilizer

[0152] The solid biological nitrogen fertilizer 1 (treatment group 1) of the present invention has significant advantages over conventional commercial organic fertilizer (control group 2) in terms of increasing yield, reducing disease, improving quality and improving soil.

[0153] In the maize experiment, compared with the control group 2, treatment group 1 showed a 14.31% increase in yield per unit area, a 58.33% increase in available phosphorus in the soil, and a 72.2% decrease in the incidence of stalk rot.

[0154] In the wheat trial, compared with the control group 2, treatment group 1 showed a 30.78% increase in yield per unit area, a 25% increase in the number of effective tillers, a 60% decrease in the incidence of sheath blight, and a 16.67% increase in thousand-grain weight.

[0155] In the cucumber experiment, compared with control group 2, treatment group 1 showed a 25.00% increase in yield per unit area, a 116.67% increase in the number of fruits per plant, a 44.29% increase in vitamin C content, and a 98% reduction in the number of Fusarium oxysporum in the soil.

[0156] 3.4 Summary

[0157] This invention quantifies and verifies the technical effects of increased yield, improved quality, reduced disease, and improved soil through planting trials on three crops: The bio-nitrogen fertilizer 1 in treatment group 1, i.e., the bio-nitrogen fertilizer provided by this invention, showed significant growth-promoting effects in the growth-promoting trials of corn, wheat, and cucumber, not only increasing yield and quality but also significantly improving soil (Table 5). This indicates that the microbial fertilizer is suitable for grain and vegetable crops, and its effect is stable on soils of different properties, improving soil properties and demonstrating universality.

[0158] Table 5. Comparison of effects between treatment group 1 and control group 2

[0159]

[0160] In summary, the soil microecological restoration bio-nitrogen fertilizer provided by this invention achieves increased yield and quality of corn, wheat, and cucumber in soils with different properties through the complementary functions of six microbial strains and rhizosphere ecological regulation, combined with the enhancement of electrochemical low-Hertz water and solid-state fermentation technology, and also plays a role in improving the soil.

Claims

1. A universal liquid compound microbial agent suitable for industrial production for soil microecological remediation, characterized in that: The following six 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; and synergistic bacteria: Trichoderma asperellum. S1: Propagate nitrogen-fixing bacteria and growth-promoting bacteria to establish a nitrogen source foundation: Add electrolytic low-hertz water to the sterilized main fermenter and add pre-mixed seed liquid of *Azotobacter spp.* and *Azotobacter brasiliensis*. 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 22-24 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 32℃, humidity of the top space of the fermenter 92%±1%, fermentation time 16-18 hours; S3: Initiate the synergistic metabolism of potassium-solubilizing bacteria and Trichoderma to activate mineral decomposition: After the fermentation of the S2 stage, 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 60%, temperature 28℃, humidity of the top space of the fermenter 88%±1%, fermentation for 34-36 hours; When the number of viable bacteria in the main fermenter is ≥2×10 10 Fermentation ends when CFU / mL is reached.

2. The universal soil micro-ecological remediation liquid compound microbial agent suitable for industrial production according to claim 1, characterized in that, The mass ratio of *Azotobacter globosum*, *Pseudomonas fluorescens*, *Bacillus mucilaginosus*, *Azotobacter brasiliensis*, *Bacillus subtilis*, and *Trichoderma echinosporum* is 3:1:1:2:1:

1.

3. The universal soil micro-ecological remediation liquid compound microbial agent suitable for industrial production according to claim 2, characterized in that, The seed culture preparation method is as follows: six 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 six 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 When CFU / mL, the seed cultures of *Azotobacter globosum* and *Azotobacter brasiliensis* were mixed at a volume ratio of 1:1 to obtain premixed seed culture 1. The seed cultures of *Pseudomonas fluorescens* and *Bacillus subtilis* were mixed at a volume ratio of 1:1 to obtain premixed seed culture 2. After incubation at 30°C for 30 minutes, the two premixed seed cultures 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 Ashube medium, the phosphate-solubilizing bacteria were prepared using KB medium, the biocontrol bacteria were prepared using LB medium, the potassium-solubilizing bacteria were prepared using potassium-solubilizing medium, and Trichoderma acicularis was prepared using PDA medium; all five media were prepared using electrochemical low-hertz water. The inoculation amounts of the premixed seed liquid 1, premixed seed liquid 2, Bacillus mucilaginosus seed liquid, and Trichoderma echinosporum seed liquid are 9.8%-10.2%, 4.8%-5.2%, 4.8%-5.2%, and 2.8%-3.2% of the fermentation volume, respectively. The inoculation amount of electrochemical low-hertz water is 76.4%-77.6% 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 universal soil micro-ecological remediation liquid compound microbial agent suitable for industrial production according to claim 3, characterized in that, During the S1 stage, when fermentation reaches the middle of the logarithmic growth phase, add the growth factors succinic acid and sodium molybdate; after the S2 stage fermentation is completed and the system pH is stable, add the growth factors glycerol and calcium phosphate; when the S3 stage is started, add the growth factor potassium feldspar powder.

5. The preparation method of the universal soil micro-ecological remediation liquid composite microbial agent suitable for industrial production according to claim 4, characterized in that, S1 succinic acid and sodium molybdate are added simultaneously and in batches using a pulsed slow-flow injection method. The concentration of succinic acid is 0.01% and the concentration of sodium molybdate is 10 μM. The addition is done in three batches, with each batch containing 1 / 3 of the total required amount, and the interval between batches is 2 hours. In S2, 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; 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 between each batch. The potassium feldspar powder in S3 is added in two batches. The potassium feldspar powder has a particle size of 200 mesh and a concentration of 10 g / L. The two additions are 6 hours apart.

6. A universal soil microecological restoration bio-nitrogen fertilizer suitable for industrial production, characterized in that, It is prepared using the universal soil microecological restoration liquid compound microbial agent suitable for industrial production as described in any one of claims 1-5.

7. The universal soil microecological restoration bio-nitrogen fertilizer suitable for industrial production 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 for a second aging and fermentation through an intelligent material distribution system: the naturally piled material is allowed to undergo deep decomposition using residual heat and residual microorganisms. Fermentation continues for 10-12 days until the end of the fermentation, thus obtaining the universal soil micro-ecological restoration bio-nitrogen fertilizer. The sign of the end of fermentation is: when the material naturally cools down to a moisture content of 15-29%, has a uniform dark brown color, loose texture, a muddy 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.

8. The universal soil microecological restoration bio-nitrogen fertilizer suitable for industrial production 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. During the stirring process, a liquid compound microbial agent is added simultaneously 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 universal soil microecological restoration bio-nitrogen fertilizer suitable for industrial production 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 universal soil microecological restoration bio-nitrogen fertilizer suitable for industrial production according to claim 9, characterized in that, The completion of the second fermentation stage A2 is indicated by the following: when the material naturally cools to a moisture content of 15-29%, the color is uniformly dark brown, the texture is loose, it has an earthy smell without other off-odors, the pH is 6.5-7.5, and the number of beneficial live bacteria is ≥2×10⁻⁶. 10 When the concentration reaches / g, the second fermentation ends.

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