High-salt-tolerance microbial liquid complex microbial inoculant suitable for saline-alkali desertification land and biological bacterial fertilizer of microbial liquid complex microbial inoculant

Abstract

The invention belongs to the technical field of biological bacterial fertilizers, and particularly relates to a high-salt-tolerance microbial liquid complex microbial inoculant suitable for saline-alkali desertification land and a biological bacterial fertilizer thereof. The microbial liquid complex microbial inoculant is prepared from the following functional strains by using electrochemical low-Hertz water through sequential inoculation and fermentation of three stages: salt-tolerant repair bacteria Nocardia halophila, salt-tolerant functional bacteria Bacillus subtilis and Azospirillum brasilense, nitrogen-fixing functional bacteria Azotobacter chroococcum, phosphorus-solubilizing functional bacteria Pseudomonas fluorescens, and nitrogen-solubilizing functional bacteria Pseudomonas fluorescens. The bacillus mucilaginosus is used as a potassium-dissolving functional bacterium, the trichoderma asperellum is used as a biocontrol growth-promoting bacterium, and the trichoderma harzianum is used as a rhizosphere optimizing bacterium. The biological bacterial fertilizer is prepared from the liquid complex microbial inoculant. Through scientific compatibility of different strains provided by the invention, efficient improvement and crop yield increase of the saline-alkali desertification land are realized, and the strain has important economic and social values.

Description

Technical Field

[0001] This invention belongs to the field of bio-fertilizer technology, specifically relating to a highly salt-tolerant microbial liquid compound agent and its bio-fertilizer suitable for saline-alkali and sandy land. Background Technology

[0002] Saline-alkali desertified land is characterized by both salinization and desertification, meaning the soil has high salinity and a large sand content, resulting in poor soil quality, poor water and fertilizer retention capacity, difficulty in vegetation growth, and a fragile ecosystem. Saline-alkali desertified land often exhibits a surface feature of mixed salt crystals and sand grains, with white salt crystals interwoven with sand grains to form a loose salt-sand layer. In arid areas, salt accumulation on the surface is significant, and a salt crust or crust may appear, hard as brick when dry and heavy and compacted when wet. Some areas may also exhibit a cracked structure, with fine sand filling the fissures, forming a unique "white, hardened soil" morphology. This type of soil typically has a flaky or vesicular-plate structure, with a crust layer 1-3 cm thick, poor permeability, and hinders crop root growth. Simultaneously, the looseness of the sandy soil exacerbates the vertical migration of salt, leading to stratified accumulation of salt and sand grains. The bottom sand layer has high permeability, while surface salt easily rises with evaporation. Saline-alkali desertification is widespread in Northwest China and is a common form of land degradation that seriously affects crop growth and yield.

[0003] Currently, my country mainly adopts four measures for the treatment of saline-alkali desertified land: First, water conservancy improvement methods, including irrigation for salt leaching, diverting fresh water to dissolve salt and draining it through a drainage system. However, in actual production, drainage after salt leaching is difficult. A large amount of fresh water is needed during the leaching process, but in many saline-alkali desertified areas, fresh water resources are scarce. Furthermore, if the drainage system is inadequate, the brine after leaching cannot be drained and will seep back into the soil. Sandy soil itself has poor water retention; after irrigation, water quickly infiltrates or evaporates, making it difficult to form a stable desalination environment. At the same time, desertification results in extremely low organic matter and clay content in the soil, making it impossible to fix salts through colloidal adsorption, leading to the loss of nutrients and soil conditioners with water. Moreover, relying solely on water is not only wasteful but also... The soil remediation methods are: 1) Severe secondary salinization, requiring annual repairs and modifications; 2) Physical remediation methods, including deep plowing and tilling to improve soil salinity and reshape soil structure, but these are complex and difficult to implement in practice; 3) Chemical remediation methods, including applying soil conditioners to lower soil pH, replace sodium ions, and increase organic fertilizer application to improve soil structure, but improving soil structure, such as increasing soil organic colloid content, is difficult and often involves large-scale operations like deep plowing, soil replacement, and irrigation / drainage facilities, resulting in significant topsoil disturbance, a high risk of wind erosion, and high costs and complex procedures requiring long-term, continuous investment to see noticeable results; 4) Biological remediation methods, including the use of salt-tolerant microbial agents to improve soil. Currently, the application of microbial agents in saline-alkali desertified land is an important research direction in soil remediation and ecological agriculture in recent years. Microbial inoculant improvement technology has advantages such as being eco-friendly and relatively low-cost. However, high salinity and alkalinity pose a severe challenge to the survival of most microbial inoculants: due to excessive soil salinity, many conventional microbial species cannot adapt, and salinity significantly damages the microbial growth environment, resulting in ineffective reproduction and colonization after application, thus greatly reducing the improvement effect. Desertification leads to extremely poor soil water and fertilizer retention capacity, with organic matter content often below 1%, failing to provide the carbon source and nutrients needed for microbial reproduction, and also failing to maintain the stable moist environment required by microorganisms. This leads to a rapid decline in the activity of inoculants, forming a vicious cycle of "weak microorganisms → low soil fertility." Severely saline-alkali soils swell and close pores after contact with water, creating an anaerobic environment, further inhibiting the function of aerobic microorganisms. Desertified soils lack clay particles and organic colloids, making it easy for microorganisms and their metabolites to be washed away with water, making it difficult to form effective colonization in the rhizosphere. Loose sand particles cannot provide a stable micro-aggregate attachment space for microorganisms, weakening their function of improving soil structure. The effectiveness of microbial agents is greatly affected by environmental factors (such as temperature, humidity, and pH), their action is relatively slow, and the effects may not be long-lasting. In areas with repeated salinization or severe secondary salinization, the improvement effect is difficult to maintain in the long term.

[0004] Furthermore, single microbial agents have limited functions, only alleviating certain types of stress (such as phosphorus solubilization, acid production, and synthesis of osmotic regulators), and cannot systematically address the complex environment of saline-alkali desertified land with multiple intertwined stresses. Compared to single agents, compound microbial agents have the core advantage of achieving functional complementarity through the synergistic effect of multiple strains, enhancing environmental adaptability and stability, and improving system repair capabilities. However, separate fermentation of each strain in a compound microbial agent leads to numerous production equipment and difficulties in production control. While mixed or partially mixed fermentation can reduce production equipment, the significant differences in culture media and cultivation conditions among different strains make mixed and partially mixed fermentation very difficult, easily resulting in problems such as microbial imbalance, functional failure or weakening, low yield, and difficulty in controlling product quality. Therefore, the industrial production of compound microbial agents faces enormous challenges, and its large-scale industrial production is prone to problems such as microbial imbalance, functional failure or weakening, low yield, and difficulty in controlling product quality. Summary of the Invention

[0005] To address the problems that existing compound microbial agents are difficult to effectively reproduce and colonize in saline-alkali desertified land after application, resulting in poor and unsustainable improvement effects, and that their industrial preparation suffers from microbial imbalance, low yield, unstable product quality, and difficulty in achieving precise optimization of production conditions, this invention provides a highly salt-tolerant liquid compound microbial agent and its bio-fertilizer suitable for saline-alkali desertified land, in order to solve the above problems.

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

[0007] A highly salt-tolerant liquid compound microbial agent suitable for saline-alkali and sandy land comprises the following functional strains, prepared using electrochemically treated low-Hertz water (small molecule cluster water) through a three-stage sequential inoculation and fermentation process: salt-tolerant restorative bacteria *Nocardia halophyte*, salt-tolerant functional bacteria *Bacillus subtilis* and *Azospirillum brasiliensis*, nitrogen-fixing functional bacteria *Azotobacter chrysogenum*, phosphate-solubilizing functional bacteria *Pseudomonas fluorescens*, potassium-solubilizing functional bacteria *Bacillus mucilaginosus*, biocontrol and growth-promoting bacteria *Trichoderma echinosporum*, and rhizosphere-optimizing bacteria *Trichoderma harzianum*. The inoculation and fermentation preparation includes the following steps:

[0008] S1: Stress-resistant foundation building period: Add electrolytic low-hertz water to the sterilized main fermenter, and add pre-mixed salt-tolerant repair bacteria and salt-tolerant functional bacteria seed liquid. Adjust the fermentation conditions to the first-order fermentation conditions: pH=7.5±0.3, dissolved oxygen content of 25-30%, temperature of 32℃±0.5℃, humidity of the top space of the fermenter of 90-92%, fermentation time of 23-25 ​​hours;

[0009] S2: Nutrient Activation Period: After the S1 stage, the conditions are adjusted to the second fermentation time. Pre-mixed seed culture of nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing bacteria is added to the main fermenter. The second fermentation time conditions are: pH=6.8±0.2, dissolved oxygen 30-40%, temperature 32℃±0.5℃, humidity of the top space of the fermenter 91-93%, and fermentation time 17-19 hours.

[0010] S3: Synergistic Sand Fixation Functional Period: After the fermentation of the S2 stage, the seed liquid of biocontrol and growth-promoting bacteria and rhizosphere optimization bacteria is directly introduced, and salt-tolerant repair bacteria seed liquid is added. Mechanical stirring is turned on and the fermentation conditions are adjusted to the third time sequence: pH=7.0±0.2, dissolved oxygen content is 40-50%, temperature is 32℃±0.5℃, humidity of the top space of the fermenter is 85-88%, and fermentation time is 35-37 hours.

[0011] When the total viable count in the main fermenter is ≥2.2×10⁻⁶ 10 Fermentation ends when CFU / mL is reached.

[0012] Preferably, the volume ratio of the seed liquid containing salt-tolerant repair bacteria: salt-tolerant functional bacteria: nitrogen-fixing functional bacteria: phosphorus-solubilizing functional bacteria: potassium-solubilizing functional bacteria: biocontrol and growth-promoting bacteria: rhizosphere-optimizing bacteria is 3:2:2:1:1:1:1.

[0013] Preferably, the end of stage S1 is marked by: the total viable count in the main fermenter ≥ 1 × 10⁻⁶. 9 CFU / mL, salt-tolerant remediation bacteria ≥55%; EC value ≤12mS / cm, salinity reduced to 1.5%; contamination rate ≤0.01%; the end of S2 stage is marked by: total viable count ≥1.5×10⁻⁶. 9 The system should have the following characteristics: CFU / mL, available phosphorus ≥ 55 mg / L, available potassium ≥ 35 mg / L, nitrogenase activity ≥ 28 nmol C2H4 / (mL·h), system viscosity ≤ 35 cP, and salinity ≤ 1.2%; the end of the S3 stage is indicated by a total viable count ≥ 2.2 × 10⁻⁶. 10 CFU / mL, with ≥2.2 × 10⁻⁶ bacteria in the viable bacteria. 10 CFU / mL, Trichoderma spores ≥2×10 7 Cells / mL; extracellular polysaccharide ≥12g / L, potassium feldspar decomposition rate ≥30%, oxalic acid content secreted by Trichoderma ≥8mmol / L; Trichoderma spore germination rate ≥92%, Bacillus mucilaginosus silicate enzyme activity ≥15U / mL; fermentation broth salinity ≤1.0%, pH stable at 7.0±0.2; sand grain binding rate ≥65%.

[0014] Preferably, the preparation and mixing of seed solutions in S1 and S2 includes pretreatment, primary seed tank culture, and secondary premix tank proportioning, with the specific steps as follows:

[0015] Step 1: Pretreatment: Each functional strain was inoculated into its corresponding specialized culture medium and cultured at 30℃ for 24-48 hours until all strains reached the logarithmic growth phase. The salt-tolerant repair bacteria were cultured on LB medium + 0.3% NaCl + 0.02% betaine; the salt-tolerant functional bacteria were cultured on LB medium + 0.3% NaCl + 0.2% proline; the biocontrol and growth-promoting bacteria were cultured on PDA medium + 0.1% arabinose; the nitrogen-fixing functional bacteria were cultured on Assambe medium; the phosphorus-solubilizing functional bacteria were cultured on KB medium + 0.1% calcium phytate + 0.01% MnSO4; the potassium-solubilizing functional bacteria were cultured on basal medium + 2% potassium feldspar powder; and the rhizosphere optimization bacteria were cultured on PDA medium + 0.15% humic acid. All specialized culture media were prepared with electrolytic low-Hertz water and sterilized by autoclaving at 121℃ for 30 minutes.

[0016] Step 2: Primary Seed Tank Culture: Configure 8 independent primary seed tanks, and add the strains cultured in Step 1 into different independent primary fermenters for culture and propagation.

[0017] Step 3: Proportional mixing in secondary premixing tanks: Two premixing tanks were selected based on stress resistance synergy and nutrient dependence to premix the strain seed liquids in two groups. One premixing tank mixed the seed liquids of Haloxylon ammodendron, Bacillus subtilis, and Azotobacter brasiliensis at a volume ratio of 3:1:1, and kept at 32°C and 90-92% humidity at the top of the fermenter for 30 minutes. The other premixing tank mixed the seed liquids of Azotobacter chrysogenum, Pseudomonas fluorescens, and Bacillus mucilage at a volume ratio of 2:1:1, and kept at 32°C and 91-93% humidity at the top of the fermenter for 20 minutes. The premixing tanks were made of 316L stainless steel and equipped with a double-helix agitator, pH / humidity sensor, and sterile feeding port.

[0018] Preferably, during stage S1, when fermentation reaches the middle of the logarithmic growth phase, the nutrients 0.05% betaine, 10 μM sodium molybdate, and 0.03% proline are added; during stage S2, 2 hours after the end of stage S1, the nutrients 2 g / L food-grade calcium phytate, 5 g / L potassium feldspar powder, and 0.02% 99% pure glutamic acid are added; during stage S3, 4 hours after seed liquid inoculation, the nutrients 0.2% 70% pure weathered coal-derived humic acid, 0.1% food-grade xanthan gum, and trace element solution are added; the trace element solution includes 0.001% Fe. 2+ and 0.0005%Mn 2+ .

[0019] Preferably, the method of adding betaine, sodium molybdate and proline in S1 is as follows: first, betaine is injected through a sterile peristaltic pump with an accuracy of ±0.1 mL / min, and then the mixture of sodium molybdate and proline is injected synchronously after an interval of 30 seconds;

[0020] The nutrient factor addition method in S2 is as follows: after mixing calcium phytate and glutamic acid, it is injected synchronously through the annular liquid distributor at the bottom of the tank with a 0.5mm aperture. After 20 minutes, potassium feldspar powder and electrolytic low-hertz water are mixed to form a 10% suspension, which is injected through the central tube of the stirring shaft that extends 10cm below the liquid surface. At the same time, the stirring speed is increased from 160r / min to 180r / min. Calcium phytate needs to be added continuously in 3 batches, with an interval of 4 hours between each batch. Glutamic acid and calcium phytate are mixed and injected synchronously through the annular liquid distributor at the bottom of the tank.

[0021] The method of adding nutrients in S3 is as follows: after mixing the three into a sterile solution, add the solution in the form of atomized liquid through the atomizing nozzle on the top of the tank. The atomization covers ≥90% of the cross section inside the tank, and the stirring speed is increased to 200r / min.

[0022] This invention also provides a highly salt-tolerant bio-fertilizer suitable for saline-alkali desertified land, which is prepared using the aforementioned highly salt-tolerant microbial liquid compound agent suitable for saline-alkali desertified land.

[0023] The method for preparing the bio-fertilizer includes:

[0024] A1: The aforementioned microbial liquid compound inoculant, lignite, wheat bran, soybean meal, and electrolytic low-hertz water 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 for the first solid-state fermentation. The solid-state fermentation system automatically adjusts the temperature during the fermentation process through a temperature sensor linked to the intermittent micro-burst oxygen frequency: the initial stage temperature is 42-45℃, maintained for 22-24 hours; the peak stage temperature is 60-65℃, maintained for 46-48 hours; and the final stage temperature is 40-45℃, maintained for 22-24 hours.

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

[0026] Preferably, the end of the first solid-state fermentation in stage A1 is marked by the following: the temperature is maintained at the final stage temperature for 24 consecutive hours, the material humidity is ≤40%, the material color is dark brown, there is no odor, it is loose when squeezed, and there is no stickiness; the end of the second aging fermentation in stage A2 is marked by the following: the material naturally dehumidifies 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 6.5-7.5, and the number of beneficial live bacteria is ≥200 million / g.

[0027] Preferably, in A1, the total amount of solid components—80-100 mesh 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 initial moisture content of the lignite powder is 10-15%, and the initial moisture content of the 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, 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 mixture reaches 50-60%.

[0028] Beneficial effects

[0029] This invention provides a highly salt-tolerant microbial liquid compound inoculant and its bio-fertilizer suitable for saline-alkali desertified land. Through a time-sequential fermentation design, it organically combines functional strains with different fermentation conditions, exhibiting "symbiotic growth and complementary growth." This combination addresses the key pain points of traditional compound inoculants applied to saline-alkali desertified land: "difficult colonization, weak reproduction, and lack of sustainability," as well as the problem of poor improvement effects. Specifically, the organic combination of functional strains innovatively adopts a four-pronged strategy: "dual pioneer colonization + metabolic closed loop + stress resistance enhancement + ecological synergy." Firstly, the dual pioneer colonization strategy utilizes the salt-tolerant remediation bacterium *Haloxylon ammodendron* as a salt-tolerant pioneer species. Its cell wall naturally synthesizes osmolarity-regulating substances such as betaine and tetrahydropyrimidine, enabling it to initiate metabolism and secrete extracellular substances even in extreme environments with NaCl concentrations as high as 15%. Polysaccharides (EPS) initially adhere to sand grains, creating a micro-territory shelter for subsequent microbial communities. The hyphal network of the biocontrol-promoting fungus Trichoderma echinosporum and the rhizosphere-optimizing fungus Trichoderma harzianum acts like "bio-glue" to penetrate the sand layer. The chitinases they produce can decompose residual organic debris in the wind and sand, rapidly releasing carbon and nitrogen sources. At the same time, they entangle loose sand grains into aggregates, forming a physical protective structure on the 0-10cm surface layer to prevent subsequent microbial species from being stripped away by wind and sand. This synergistic model of "bacterial biofilm formation - fungal sand fixation" increases the initial colonization success rate of microbial communities from less than 5% with single-strain application to over 60%. Simultaneously, a self-sufficient metabolic closed loop of "nitrogen self-supply - phosphorus and potassium activation - growth induction" is constructed. Relying on the synergistic effect of nitrogen-fixing functional bacteria such as Azotobacter chrysogenum, phosphorus-solubilizing functional bacteria such as Pseudomonas fluorescens, potassium-solubilizing functional bacteria such as Bacillus mucilaginosus, and salt-tolerant functional bacteria such as Azotobacter brasiliensis, the bacterial community can still reproduce exponentially in sand layers lacking organic matter, completely solving the problem of rapid decline of traditional bacterial agents due to carbon source depletion. The microbial community's resilience is further enhanced through biofilms and signal transduction systems: Bacillus subtilis, a salt-tolerant functional bacterium, secretes lipopeptide surfactants to reduce the surface tension of the bacteria, helping other strains to adhere stably to the salt crystal surface; Pseudomonas fluorescens synthesizes siderophores to chelate scarce iron elements, while inhibiting the growth of pathogens, providing a clean colonization interface for Trichoderma echinosporum and Trichoderma harzianum; Trichoderma echinosporum and Trichoderma harzianum form a three-dimensional network of hyphal matrix, embedding functional bacteria within it, making the pH of the microenvironment inside the biofilm 1.5-2 units lower than that outside the membrane, reducing the salt concentration by 40%-60%, and significantly alleviating high salt stress.In addition, the microbial community also forms a synergistic strategy of "niche displacement + nutrient activation": In terms of niche displacement, Haloxylon ammodendron, Trichoderma echinosporum, and Trichoderma harzianum mainly colonize non-rhizosphere barren sand layers, avoiding competition with native fungi for limited rhizosphere nutrients; functional fungi such as Azotobacter chrysogenum preferentially occupy the surface of newly formed soil aggregates, where the abundance of native fungi is extremely low and the competitive pressure is small; in terms of nutrient activation, Bacillus mucilaginosus decomposes potassium feldspar and releases silicon, which can stimulate the germination of native arbuscular mycorrhizal fungi (AMF); Bacillus subtilis degrades complex organic matter that is difficult for native fungi to utilize, and its metabolites become a high-quality carbon source for native actinomycetes, ultimately forming a stable community of "external fungi-native fungi" synergistic symbiosis. To ensure long-lasting improvement effects, the various strain combinations construct a three-tiered dormancy system of "hyphae-spores-cysts": *Trichoderma echinosporum* and *Trichoderma harzianum* produce chlamydospores under harsh conditions, which can survive for 2-3 years; *Bacillus mucilaginosus* and *Bacillus subtilis* form stress-resistant spores that can withstand high temperatures of 50°C and UV radiation; and *Haloxylon ammodendron* produces hyphal fragments and extracellular vesicles, which act as "resurrection seeds" dormant in the sand layer. When rainfall or irrigation occurs, these dormant bodies can quickly revive, achieving "one application, effective for many years." Field trials show that this strain combination can still maintain more than 60% of the initial colonization density in the second year after application, while the retention rate of ordinary inoculants is only 5%-10% during the same period. In addition to its core function of improving soil quality, the microbial community also has multiple synergistic effects: *Pseudomonas fluorescens* secretes organic acids to dissolve insoluble phosphorus (such as Ca3(PO4)2) in saline-alkali desertified land, thus both obtaining phosphorus and helping to lower soil pH; *Azospirillum brasiliensis* relies on phosphorus produced by *Pseudomonas fluorescens* to synthesize IAA, thereby strengthening crop growth regulation under saline-drought stress; *Bacillus subtilis* proliferates in large quantities in the microenvironment formed by *Trichoderma echinosporum* and *Trichoderma harzianum*, secreting antimicrobial peptides to inhibit pathogens in desertified land, while sharing carbon sources with *Pseudomonas fluorescens*, exhibiting strong metabolic compatibility; *Bacillus mucilaginosus* secretes silicate enzymes to activate potassium feldspar on the basis of sand fixation and salt reduction, while *Trichoderma harzianum* and *Trichoderma echinosporum* jointly secrete organic acids, which not only enhance the salt reduction effect but also decompose organic matter to improve the fertilizer retention capacity of sandy soil.

[0030] This invention addresses key challenges commonly encountered in the industrial production of compound microbial agents, such as microbial imbalance, low yield, and quality fluctuations, through precise design of two-stage premixing and three-stage main fermentation timing conditions, achieving stable and controllable production processes. Two independent premixing tanks are used to pre-adapt the strains to different groups, avoiding stress reactions caused by direct introduction of different functional microbial communities into the main tank: the stress-resistant premixing tank mixes *Nocardia halophyte*, *Bacillus subtilis*, and *Azotobacter brasiliensis* seed liquid in a 3:1:1 volume ratio and incubates at 32°C and 90-92% humidity for 30 minutes. Its core function is to activate salt-tolerant genes and construct a low-salt adaptation system, ensuring rapid formation of a stress-resistant dominant population after introduction into the main tank. The nutrient activation premixing tank mixes *Azotobacter chrysogenum*, *Pseudomonas fluorescens*, and *Bacillus mucilaginosus* seed liquid in a 2:1:1 volume ratio and incubates at 32°C and 91-93% humidity for 20 minutes. Its core function is to coordinate nutrient metabolism pathways, laying the groundwork for efficient phosphorus and potassium activation functions in the subsequent process.

[0031] This invention employs a three-stage dynamic control system for the main fermenter: time-series adaptation and progressive functional enhancement. Stage S1 focuses on "salt tolerance, salt reduction, and basic nitrogen supply." This stage selects a stress-resistant basic population based on precise matching of strain characteristics: Haloxylon ammodendron requires a low-salt microenvironment (reducing from an initial 3% to below 1.5%) to provide suitable growth conditions for Bacillus subtilis and Azotobacter brasiliensis. All three are medium-to-slow-growth strains; a microaerobic environment avoids the inhibition of nitrogenase activity in Azotobacter brasiliensis by high dissolved oxygen, while high humidity meets the needs of Haloxylon ammodendron for secreting extracellular polysaccharides. If inoculated simultaneously with the nutrient-activating strains, the growth of Haloxylon ammodendron would be suppressed by the rapidly proliferating bacteria, resulting in a 70% decrease in salt reduction rate. Therefore, it needs to be premixed separately and introduced preferentially. Ultimately, through the synergy of these three strains, a stable stress-resistant basic population is formed within the main fermenter, creating a low-salt, sterile microenvironment for subsequent microbial inoculation. Stage S2 focuses on the "nitrogen, phosphorus, and potassium activation" function. This stage incorporates a nutrient activation group and relies on the low-salt environment and basic nitrogen source accumulated in Stage S1: *Azotobacter globosum* continuously fixes nitrogen, providing nitrogen support for *Pseudomonas fluorescens* to synthesize phytase and *Bacillus mucilaginosus* to synthesize silicate enzymes; a slightly acidic environment can increase phytase activity by 30%, and high dissolved oxygen meets the aerobic metabolic needs of *Pseudomonas fluorescens*. If this stage is started prematurely, the activation rates of effective phosphorus and potassium will decrease by 85% and 76%, respectively, due to the lack of a low-salt environment and nitrogen source. Through regulation in this stage, the effective phosphorus in the main tank is ≥55 mg / L and the effective potassium is ≥35 mg / L, providing sufficient nutrients for the sand-fixing bacteria in Stage S3. Stage S3 focuses on the "sand fixation-efficiency enhancement-stress resistance strengthening" function. The design logic is: *Bacillus mucilaginosus* needs to proliferate first to form a population advantage (≥8×10⁻⁶). 9The concentration of *Trichoderma* spores (CFU / mL) is reduced to prevent inhibition by *Trichoderma* hyphae. *Trichoderma echinosporum* and *Trichoderma harzianum* are strictly aerobic fungi; high dissolved oxygen and medium humidity promote spore germination (germination rate ≥92%) and extracellular polysaccharide synthesis (≥12 g / L). The added *Haloxylon ammodendron* can continuously reduce salinity (ultimately ≤1.0%), synergistically enhancing the salinity-reducing effect with the organic acids secreted by *Trichoderma*. If introduced simultaneously, the spore germination rate of *Trichoderma echinosporum* is only 55%, extracellular polysaccharide production decreases by 60%, and the sand-fixing effect is significantly reduced.

[0032] The entire fermentation process relies on a "premixed adaptation - sequential connection - metabolic complementarity" design to achieve efficient operation: In the S1 stage, Haloxylon ammodendron provides a low-salt environment for all subsequent bacterial groups; in the S2 stage, the nitrogen source from Azotobacter chrysogenum supports the mycelial growth of Trichoderma harzianum in the S3 stage; and in the S3 stage, the citric acid secreted by Trichoderma harzianum supplements the carbon source for the remaining Azotobacter brasiliensis in the S1 stage. The surfactants produced by Bacillus subtilis enhance biofilm permeability, increasing the enzyme production efficiency of Trichoderma harzianum by 35%. No additional culture medium or nitrogen source is required; the growth needs of all bacterial species are met solely through electrochemical low-Hertz water and the metabolic complementarity of the bacterial strains. Simultaneously, the synergistic effect of putrescine and spermidine production by Bacillus subtilis and the acid production by Trichoderma harzianum and Trichoderma harzianum maintains system pH fluctuations ≤0.3, and potassium silicate-induced K... + The expression level of the efflux gene (SKC1) was increased by 3.2 times, which enabled the bacterial community to survive for ≥80% in a 15% NaCl environment, ensuring that each strain could function normally.

[0033] Furthermore, all three fermentation sequences used electrochemically low-Hertz water as the substrate. Its unique physicochemical properties provide multiple benefits to microbial metabolism: its permeability is more than 30% higher than ordinary water, and its water molecule clusters are only 0.5 nanometers in diameter, allowing for rapid passage through cell membrane water channels, accelerating nutrient intake and metabolic waste excretion; it is weakly alkaline (pH 7.5-8.0), neutralizing acidic metabolites such as lactic acid and acetic acid produced during fermentation, maintaining the optimal pH environment for enzymatic reactions; it also enhances the permeability of microbial cell membranes, promotes the transmission of quorum sensing signal molecules, optimizes the metabolic network of the complex microbial community, and improves cell membrane stability, enhancing the survival rate of the inoculum under high temperature and high salt stress conditions. In summary, electrochemically low-Hertz water, with its unique advantages, significantly improves microbial metabolic efficiency and functional product synthesis capabilities, becoming an important support for efficiency enhancement in industrial production.

[0034] During the S1 stage, when fermentation reaches the mid-logarithmic growth phase, nutrient factors are added. This is the optimal window for nutrient fortification, as the bacterial metabolic activity is at its highest at this time. The added ingredients are 0.05% (w / v) betaine (99.2% purity), 10μM sodium molybdate (99.5% purity), and 0.03% proline (98.8% purity). Betaine, as a highly efficient osmotic protectant, can specifically induce the expression of salt-tolerant genes (such as proline synthase genes) in *Nocardia halophyte*, promoting the synthesis of its required osmotic regulators and enhancing its survival ability in high-salt environments. Sodium molybdate is a core cofactor of nitrogenase in *Azospirillum brasiliensis*, which can be embedded in the enzyme's active site to maintain conformational stability, increasing nitrogenase activity by more than 40% and ensuring a basic nitrogen source supply. Proline acts as an intracellular osmotic regulator, not only enhancing the stress resistance of *Nocardia halophyte* but also providing stress protection for *Bacillus subtilis*, reducing damage to the cell membrane from hyperosmotic environments. The addition method employs precise timing control: betaine is first slowly injected using a sterile peristaltic pump with an accuracy of ±0.1 mL / min. After a 30-second interval, sodium molybdate and proline are pre-mixed to prepare a sterile solution, which is then injected synchronously using the same peristaltic pump. This order of addition avoids competition between betaine and metal ions for transport protein channels on the cell membrane of Haloxylon ammodendron, ensuring efficient absorption of all nutrients.

[0035] Stage S2 occurs 2 hours after Stage S1, with the addition of nutrients. This stage ensures that the introduced nutrient-activated bacterial flora (Azotobacter chrysogenum, Pseudomonas fluorescens, and Bacillus mucilaginosus) has adapted to the main tank environment and its metabolic pathways have been initially activated. Food-grade calcium phytate (5μm particle size, excellent dispersibility) 2g / L, potassium feldspar powder (10μm particle size, silicon content ≥65%) 5g / L, and 99% pure glutamic acid 0.02% are added. Calcium phytate is a specific inducing substrate for phytase synthesis in Pseudomonas fluorescens. Its phosphate groups trigger the enzyme production signaling pathway, increasing phytase secretion by 60%, thereby efficiently decomposing insoluble phosphorus in the fermentation system and subsequent soil. The aluminosilicate structure in potassium feldspar powder induces Bacillus mucilaginosus to secrete silicate enzymes, while its porous structure adsorbs free Na+ in the fermentation broth. + It helps reduce the salinity of the system, creating a better environment for bacterial growth; glutamic acid, as a fast-acting nitrogen source, can directly participate in the enzyme protein synthesis of Pseudomonas fluorescens and Bacillus mucilaginosus, solving the problem of relatively insufficient nitrogen source in the early stage of stage II, and shortening the enzyme synthesis cycle by 25%.

[0036] In the S3 stage, nutrient factors are added 4 hours after seed culture inoculation, specifically 4 hours after inoculation with *Trichoderma harzianum* and *Trichoderma harzianum*. At this time, the fungi are in the early stage of spore germination, with hyphae just breaking through the spore wall, and urgently need nutritional support for hyphal extension and functional expression. The added nutrients include 0.2% humic acid derived from 70% pure weathered coal, 0.1% food-grade xanthan gum, and trace element solution (containing Fe). 2+ 0.001%, Mn 2+0.0005%. Humic acid derived from weathered coal is rich in humic substances and phenolic compounds, which can stimulate the spore germination receptors of *Trichoderma harzianum* and *Trichoderma harzianum*, increasing the germination rate from 65% in its natural state to 92%. Simultaneously, its carboxyl groups can react with Na+ in the soil. + Combined with other ingredients, it assists in subsequent salt reduction; xanthan gum, as a natural high-molecular polymer, can increase the viscosity of the fermentation broth to 50 mPa·s after addition. This provides an attachment carrier for Trichoderma mycelia, reducing mycelial breakage caused by stirring and shearing forces, and also encapsulates Bacillus mucilaginosus to form micro-aggregates, extending its survival time in desertified areas; the trace element solution contains Fe... 2+ It is the active center component of chitinase produced by Trichoderma harzianum, Mn 2+ This can activate the silicate enzyme activity of Bacillus mucilaginosus, and the two work together to complement the sand-fixing and mineral activation functions of the bacterial community.

[0037] The "segmented culture-group premixing-three-stage regulation" system designed in this invention combines scientific rigor with high efficiency: on the one hand, it reduces microbial stress and avoids interspecies antagonism through two-stage premixing; on the other hand, it achieves relay utilization of metabolites through sequential processing (S1 stage for salt reduction → S2 stage for fertilization → S3 stage for sand fixation), significantly improving functional expression efficiency. More importantly, the main fermenter only requires the addition of electrochemically low-Hertz water and micronutrients, eliminating the need for additional carbon and nitrogen sources, greatly simplifying the production process and effectively reducing industrial production costs. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be further described clearly and completely below in conjunction with specific embodiments of the present invention.

[0039] Unless otherwise specified, the components described in the embodiments of this invention are all readily available to those skilled in the art.

[0040] First Example: High Salt-Tolerant Microbial Liquid Compound Agent Suitable for Saline-Alkali Sandy Land

[0041] This set of embodiments provides a highly salt-tolerant liquid compound microbial agent suitable for saline-alkali and sandy land, comprising the following functional strains prepared by electrochemical low-Hertz water through three-stage sequential inoculation and fermentation: salt-tolerant restorative bacteria *Nocardia halophyte*, salt-tolerant functional bacteria *Bacillus subtilis* and *Azospirillum brasiliensis*, nitrogen-fixing functional bacteria *Azotobacter chrysogenum*, phosphate-solubilizing functional bacteria *Pseudomonas fluorescens*, potassium-solubilizing functional bacteria *Bacillus mucilaginosus*, biocontrol and growth-promoting bacteria *Trichoderma echinosporum*, and rhizosphere-optimizing bacteria *Trichoderma harzianum*. The inoculation and fermentation preparation includes the following steps:

[0042] S1: Stress-resistant foundation building period: Electrolytic low-Hertz water is added to the sterilized main fermenter, along with pre-mixed salt-tolerant repair bacteria and salt-tolerant functional bacteria seed liquid (accounting for 13.8%-14.2% of the fermentation volume). The fermentation conditions are adjusted to the first-order fermentation conditions: pH=7.5±0.3, dissolved oxygen content of 25-30%, temperature of 32℃±0.5℃, humidity of the top space of the fermenter of 90-92%, and fermentation time of 23-25 ​​hours. In a specific embodiment of the present invention, the seed liquid is inoculated at a ratio of 3:1:1 of Haloxylon ammodendron: Bacillus subtilis: Azotobacter brasiliensis seed liquid, and the following nutrient factors and parameters are added: 0.05% (w / v) betaine (99.2% purity), 10μM sodium molybdate (99.5% purity), and 0.03% proline (98.8% purity). Betaine, as a highly efficient osmotic protectant, can specifically induce the expression of salt-tolerant genes (such as proline synthase genes) in Haloxylon ammodendron, promoting the synthesis of osmotic regulatory substances required by the bacteria and enhancing their survival ability in high-salt environments. Sodium molybdate is a core cofactor of nitrogenase in Azotobacter brasiliensis, which can be embedded in the enzyme's active site to maintain conformational stability, thereby increasing nitrogenase activity by more than 40% and ensuring the supply of basic nitrogen sources. Proline can serve as an intracellular osmotic regulator, which can not only enhance the stress resistance of Haloxylon ammodendron but also provide stress protection for Bacillus subtilis, reducing the damage to the bacterial cell membrane caused by hyperosmotic environments.

[0043] Timing of addition: 14 hours after fermentation (2 hours before the peak of the logarithmic growth phase, when the metabolic activity of the cells is at its highest).

[0044] Addition method: First, slowly inject betaine using a sterile peristaltic pump with an accuracy of ±0.1 mL / min. After a 30-second interval, pre-mix sodium molybdate and proline to prepare a sterile solution, and then inject it synchronously using the same peristaltic pump. This addition sequence avoids competition between betaine and metal ions for transport protein channels on the cell membrane of Haloxylon ammodendron, ensuring efficient absorption of various nutrients.

[0045] The end of this stage is marked by: a total viable count ≥ 1 × 10⁻⁶ in the main fermenter. 9 CFU / mL, Haloxylon ammodendron ≥55%; EC value ≤12mS / cm, salinity reduced to 1.5%; contamination rate ≤0.01%.

[0046] S2: Nutrient Activation Period: After the S1 stage, the fermentation conditions are adjusted to the second time sequence. Pre-mixed nitrogen-fixing bacteria (Azotobacter chrysogenum), phosphorus-solubilizing bacteria (Pseudomonas fluorescens), and potassium-solubilizing bacteria (Bacillus mucilaginosus) are added to the main fermenter (accounting for 8.3%-8.7% of the fermentation volume). The second time sequence fermentation conditions are: pH=6.8±0.2, dissolved oxygen 30-40%, temperature 32℃±0.5℃, humidity at the top of the fermenter 91-93%, and fermentation time 17-19 hours. In a specific embodiment of the invention, nitrogen-fixing bacteria, phosphorus-solubilizing bacteria, and potassium-solubilizing bacteria are inoculated at a seed-liquid volume ratio of 2:1:1, and the following nutrient factors and parameters are added: food-grade calcium phytate (particle size 5μm, excellent dispersibility) 2g / L, potassium feldspar powder (particle size 10μm, silicon content ≥65%) 5g / L, and 99% pure glutamic acid 0.02%. Calcium phytate is a specific inducing substrate for phytase synthesis in *Pseudomonas fluorescens*. The phosphate groups in its molecular structure trigger the enzyme production signaling pathway, increasing phytase secretion by 60%, thereby efficiently decomposing insoluble phosphorus in the fermentation system and subsequent soil. The aluminosilicate structure in potassium feldspar powder can induce *Bacillus mucilaginosus* to secrete silicate enzymes, while its porous structure can adsorb free Na+ in the fermentation broth. + It helps to reduce the salinity of the system, creating a better environment for bacterial growth; glutamic acid, as a fast-acting nitrogen source, can directly participate in the enzyme protein synthesis of Pseudomonas fluorescens and Bacillus mucilaginosus, solving the problem of relatively insufficient nitrogen source in the early stage and shortening the enzyme synthesis cycle by 25%.

[0047] Timing of addition: 2 hours after the end of the S1 stage, when the total fermentation time is 26 hours. This point ensures that the introduced nutrient-activating bacteria (Azotobacter chrysogenum, Pseudomonas fluorescens, Bacillus mucilaginosus) have adapted to the main tank environment and the metabolic pathways have been initially activated.

[0048] Addition method: After mixing calcium phytate and glutamic acid, inject them simultaneously through a 0.5mm aperture annular distributor at the bottom of the tank. After a 20-minute interval, inject a 10% suspension of potassium feldspar powder and electrolytic low-Hertz water through the central tube of the stirring shaft, which extends 10cm below the liquid surface, while simultaneously increasing the stirring speed from 160r / min to 180r / min to prevent substrate sedimentation. Calcium phytate needs to be added in three consecutive batches (total dosage 2g / L, 4-hour intervals). Glutamic acid (total dosage 0.02%) is mixed with each batch of calcium phytate in the corresponding proportion and injected simultaneously through the annular distributor at the bottom of the tank. Calcium phytate has a fine particle size, and adding it all at once can easily lead to localized precipitation. Glutamic acid is a fast-acting nitrogen source, and adding it in batches can avoid a sudden increase in nitrogen concentration that inhibits the activity of phosphate-solubilizing bacteria. The simultaneous addition of both in batches can precisely match the metabolic needs of the bacterial community, ensuring continuous and efficient synthesis of phytase.

[0049] The end of this stage is marked by a total viable count ≥ 1.5 × 10⁻⁶. 9CFU / mL, available phosphorus ≥55mg / L, available potassium ≥35mg / L, nitrogenase activity ≥28nmol C2H4 / (mL·h), system viscosity ≤35cP, salinity ≤1.2%.

[0050] S3: Synergistic Sand-Fixing Functional Period: After the S2 stage fermentation is completed, directly inoculate with Trichoderma echinococcus and Trichoderma harzianum seed liquids (each accounting for 2.8%-3.2% of the fermentation volume) and supplement with Haloxylon ammodendron (accounting for 1.4%-1.6% of the fermentation volume). Start mechanical stirring and adjust to the third-sequence fermentation conditions: pH=7.0±0.2, dissolved oxygen content of 40-50%, temperature of 32℃±0.5℃, humidity of the top space of the fermenter of 85-88%, fermentation for 35-37 hours; the mixed Trichoderma echinococcus and Trichoderma harzianum liquid and the supplemented Haloxylon ammodendron seed liquid should meet the requirement that the spore density of the Trichoderma seed liquid is ≥1×10 7 Spores / mL, density of supplemented halophyte Nocardia seed culture ≥5×10 8 CFU / mL, initial fungal spore count ≥6×10⁻⁶ after inoculation. 4 Given the spore / mL ratio (adapted to subsequent germination needs), no adjustment is required. Add the following nutrients with the following parameters: 0.2% 70% pure weathered coal-derived humic acid, 0.1% food-grade xanthan gum, and trace element solution (containing Fe). 2+ 0.001%, Mn 2+ 0.0005%); Humic acid from weathered coal is rich in humic substances and phenolic compounds, which can stimulate the spore germination receptors of Trichoderma acicularis and Trichoderma harzianum, increasing the germination rate from 65% in the natural state to 92%. Simultaneously, its carboxyl groups can react with Na+ in the soil. + Combined with other ingredients, it assists in subsequent salt reduction; xanthan gum, as a natural high-molecular polymer, can increase the viscosity of the fermentation broth to 50 mPa·s after addition. This provides an attachment carrier for Trichoderma mycelia, reducing mycelial breakage caused by stirring and shearing forces, and also encapsulates Bacillus mucilaginosus to form micro-aggregates, extending its survival time in desertified areas; the trace element solution contains Fe... 2+ It is the active center component of chitinase produced by Trichoderma harzianum, Mn 2+ This can activate the silicate enzyme activity of Bacillus mucilaginosus, and the two work together to complement the sand-fixing and mineral activation functions of the bacterial community.

[0051] Timing of addition: 48 hours after inoculation with Trichoderma acicularis and Trichoderma harzianum. At this time, the fungus is in the early stage of spore germination, and the hyphae have just broken through the spore wall. It urgently needs nutritional support for hyphal extension and functional expression.

[0052] Addition method: After mixing the three ingredients to form a sterile solution, atomize it simultaneously through the atomizing nozzle on the top of the tank (droplet diameter 10μm), with the atomization covering ≥90% of the tank's internal cross-section. At the same time, increase the stirring speed to 200r / min to ensure that the atomized liquid and fermentation liquid blend for ≤3min.

[0053] The end of this stage is marked by: ① Total viable bacteria count ≥ 2.2 × 10⁻⁶ 10 CFU / mL (of which bacteria ≥ 2.2 × 10⁻⁶) 10 CFU / mL, Trichoderma spores ≥2×10 7 ① Count / mL; ② Extracellular polysaccharide ≥12g / L, potassium feldspar decomposition rate ≥30%, oxalic acid content secreted by Trichoderma ≥8mmol / L; ③ Trichoderma spore germination rate ≥92%, Bacillus mucilaginosus silicate enzyme activity ≥15U / mL; ④ Fermentation broth salinity ≤1.0%, pH stable at 7.0±0.2; ⑤ Sand grain binding rate ≥65%.

[0054] In a specific embodiment of the present invention, the amount of electrochemical low-hertz water introduced is 69-71% of the fermentation volume, the electrochemical low-hertz water has a molecular cluster of ≤6 molecules, and the pH is 7.0–7.5.

[0055] In a specific embodiment of the present invention, the volume ratio of the salt-tolerant repair bacteria: salt-tolerant functional bacteria: nitrogen-fixing functional bacteria: phosphorus-solubilizing functional bacteria: potassium-solubilizing functional bacteria: biocontrol and growth-promoting bacteria: rhizosphere-optimizing bacteria seed liquid is 3:2:2:1:1:1:1.

[0056] In a specific embodiment of the present invention, the preparation and pre-mixing of the seed solution in S1 and S2 includes: pretreatment, primary seed tank culture, and secondary premix tank proportional mixing, with the specific steps as follows:

[0057] Step 1: Strain pretreatment. Each functional strain was inoculated into a dedicated culture medium and incubated at 32℃ for 24-48 hours until the logarithmic growth phase. All dedicated culture media were prepared with electrolytic low-hertz water and autoclaved at 121℃ for 30 minutes. Specifically: the salt-tolerant repair bacteria were cultured on LB medium + 0.3% NaCl + 0.02% betaine; the salt-tolerant functional bacteria were cultured on LB medium + 0.3% NaCl + 0.2% proline; the biocontrol and growth-promoting bacteria were cultured on PDA medium + 0.1% arabinose; the nitrogen-fixing functional bacteria were cultured on Assumption medium; the phosphorus-solubilizing functional bacteria were cultured on KB medium + 0.1% calcium phytate + 0.01% MnSO4; the potassium-solubilizing functional bacteria were cultured on basal medium + 2% potassium feldspar powder; and the rhizosphere optimization bacteria were cultured on PDA medium + 0.15% humic acid.

[0058] Step 2: Primary Seed Tank Cultivation: Configure 8 independent primary seed tanks, add the strains cultivated in Step 1 into different independent primary fermenters, and propagate them separately. The cultivation conditions and parameters are shown in Table 1.

[0059] Table 1. Culture conditions and parameters for 8 strains

[0060]

[0061] Step 3: Proportional mixing in two premixing tanks: Two premixing tanks were selected according to the stress resistance synergy and nutrient dependence to premix the bacterial seed liquids of the strains. One premixing tank mixed the seed liquids of Haloxylon ammodendron: Bacillus subtilis: Azotobacter brasiliensis in a volume ratio of 3:1:1 to obtain the stress-resistant basic group bacterial liquid; the other premixing tank mixed the seed liquids of Azotobacter chrysogenum: Pseudomonas fluorescens: Bacillus mucilaginosus in a volume ratio of 2:1:1 to obtain the nutrient-activated group bacterial liquid. The strains and their culture conditions for the premixing groups corresponding to the two premixing tanks are shown in Table 2. The premixing tanks are made of 316L stainless steel and are equipped with a double helical agitator, pH / humidity sensor and sterile feeding port.

[0062] Table 2. Strains and culture conditions of the premixed groups corresponding to the two premixing tanks

[0063]

[0064] In a specific embodiment of the present invention, after the above process is completed, 70% of the fermentation volume of small molecule cluster water is added to the main fermenter, and fermentation is completed sequentially. In stage S1, 14% of the stress-resistant basic bacterial solution is added, and the pH is controlled at 7.5±0.3, dissolved oxygen at 25-30%, temperature at 32℃, and humidity at the top of the fermenter at 90-92%. After 14 hours of fermentation, 0.05% betaine is added first, followed by 10μM sodium molybdate and 0.03% proline 30 seconds later. At the end of fermentation, the total bacterial count is ≥1.0×10⁻⁶. 9 CFU / mL, salt reduction rate ≥18%. In the S2 stage (24-42h), inoculate with 8.5% nutrient-activated bacterial solution, adjusting to pH 6.8±0.2, dissolved oxygen 30-40%, temperature 32℃, and humidity of the fermenter top space 91-93%. At 26h of fermentation, calcium phytate needs to be added in three consecutive additions (total dosage 2g / L, 4-hour intervals). Glutamic acid (total dosage 0.02%) is mixed with the calcium phytate in the corresponding proportions. After a 20-minute interval, a 10% suspension made of potassium feldspar powder and electrolytic low-Hertz water is injected through the central tube of the stirring shaft, 10cm below the liquid surface, while simultaneously increasing the stirring speed from 160r / min to 180r / min to prevent substrate sedimentation. The total bacterial count at the end is ≥1.5×10⁻⁶. 9CFU / mL, available phosphorus ≥45mg / L. In stage S3, Trichoderma and Haloxylon ammodendron (total percentage 7.5%) were added, and the pH was adjusted to 7.0±0.2, dissolved oxygen 40-50%, fermenter top temperature 30℃, and humidity 85-88%. Humic acid, xanthan gum, and trace element solution were added via atomization after 48 hours of fermentation, and the stirring speed was increased to 200r / min. At the end of fermentation, the total viable count was ≥2.2×10⁻⁶. 10 CFU / mL, potassium feldspar decomposition rate ≥30%.

[0065] Second set of examples: High salt-tolerant bio-fertilizer suitable for saline-alkali and sandy land

[0066] This set of embodiments provides a highly salt-tolerant bio-fertilizer suitable for saline-alkali and sandy land, prepared using the microbial liquid compound inoculant described in the first set of embodiments.

[0067] In a specific embodiment of the present invention, the method for preparing the bio-fertilizer includes:

[0068] A1: The aforementioned microbial liquid compound inoculant, lignite, wheat bran, soybean meal, and electrolytic low-Hertz water are added to an intelligent mixing system. After uniform mixing, the mixture is conveyed to an intelligent solid-state fermentation system production line via a self-invented intelligent material distribution system (patent authorization announcement number CN116553963B) for the first solid-state fermentation. The intelligent fermentation system regulates the temperature during fermentation through a temperature sensor and a linked intermittent micro-burst oxygen frequency: the initial stage temperature is 42-45℃, maintained for 22-24 hours; the peak stage temperature is 60-65℃, maintained for 46-48 hours; and the final stage temperature is 40-45℃, maintained for 22-24 hours. The temperature remains constant for 24 consecutive hours, and the material moisture content remains relatively stable. When the content of solid-state fermentation reaches ≤40%, the material is dark brown in color, odorless, loose when squeezed, and without clumping, the first solid-state fermentation is complete. In A1, the total amount of solid components is 100%: 80-100 mesh lignite powder, wheat bran, and soybean meal. The proportions of each solid component are: lignite powder 70-85%, wheat bran 8-15%, and soybean meal 8-20%. The initial moisture content of lignite powder is 10-15%, and the initial moisture content of wheat bran and soybean meal is 8-12%. After the above solid components are mixed evenly, electrolytic low-hertz water is added and stirred thoroughly. At the same time, liquid compound microbial agent is added simultaneously during the stirring process, with the addition amount calculated as 0.8-10% of the total solid components, so that the total moisture content of the mixture reaches 50-60%.

[0069] A2: The material obtained from the first solid-state fermentation in A1 is rearranged into the aging and fermentation production line through an intelligent material distribution system for the second aging and fermentation: The material is naturally piled up, and combined with intelligent monitoring, the residual heat and residual microorganisms of the material are used to complete the deep decomposition, which lasts for 10-12 days. When the material naturally cools down to a moisture content of 15-29%, the color is uniformly dark brown, the texture is loose, it has an earthy smell without other odors, the pH is stable at 6.5-7.5, and the number of beneficial live bacteria is ≥200 million / g, the second aging and fermentation is completed.

[0070] Experimental Example

[0071] Experimental Example 1. Effects of Electrochemical Low-Hertz Water on the Number of Viable Cells at Different Stages of Fermentation

[0072] Experimental group: Microbial liquid compound inoculant prepared according to the method in the first group of examples.

[0073] Control group: The electrochemical low-hertz water in the first embodiment was replaced with sterilized water, and all other operations were the same as in the first embodiment.

[0074] The viable cell counts at each fermentation stage were measured in both the experimental and control groups. Statistical analysis was performed using ANOVA (α=0.05), with at least three replicates per group. The results are shown in Table 3. At the end of the first time step, the viable cell count in the experimental group was 73.6% higher than that in the control group; at the end of the second time step, the viable cell count in the experimental group was 76.2% higher than that in the control group; at the end of the third time step, the viable cell count in the experimental group was 85.7% higher than that in the control group; after 3 months of storage at room temperature, the viable cell count in the experimental group was 93.9% higher than that in the control group. This indicates that electrochemical low-hertz water can effectively improve fermentation efficiency.

[0075] Table 3. Effects and mechanisms of electrochemical low-Hertz water on viable bacterial count

[0076]

[0077] Experimental Example 2. Effects of High Salt Tolerance Bio-fertilizer

[0078] 2.1 Rose Experiment in Alashan Desertified Saline-Alkali Land, Inner Mongolia

[0079] (1) Test fertilizers and fertilization methods:

[0080] Processing Group 1:

[0081] Fertilizer: The bio-fertilizer 1 prepared according to the preparation method in the second set of examples has a liquid compound microbial agent added at a rate of 10%.

[0082] Application method: Use Luyuan commercial organic fertilizer (800 kg / mu) as base fertilizer, apply bio-fertilizer 1 (15 kg / mu) in the planting hole at the time of transplanting, and apply bio-fertilizer 1 once in each planting hole during the budding and full bloom stages, with the amount of bio-fertilizer 1 used each time being 16 kg / mu; spray bio-fertilizer 1 once in each of the budding and post-flowering stages (3 kg / mu).

[0083] Processing Group 2:

[0084] Fertilizer: Replace the electrochemical low-hertz water in the preparation method of the second group embodiment with ordinary sterilized water, and the other operations are exactly the same as in the second group embodiment to obtain bio-fertilizer 2;

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

[0086] Control group 1:

[0087] Fertilizer: Replace the liquid compound bacterial agent in the preparation method of the second group of examples with a single halophyte Nocardia, and the other operations are exactly the same as in the second group of examples.

[0088] Application method: Same as treatment group 1.

[0089] Control group 2:

[0090] Fertilizers: Sinochem compound fertilizer (15-15-15), Luyuan commercial organic fertilizer and Sinochem high phosphorus compound fertilizer (15-45-15);

[0091] Application method: Use Sinochem compound fertilizer (45kg / mu) and Luyuan commercial organic fertilizer (800kg / mu) as base fertilizer. Use Sinochem high phosphorus compound fertilizer (15kg / mu) at the time of transplanting. Apply top dressing once each during the budding stage, budding stage, full bloom stage and post-flowering stage, with each application of Sinochem compound fertilizer at 20kg / mu.

[0092] (2) Test location:

[0093] In the Alashan Rose Sandy Saline Soil Experimental Area, the sand grain content (0.05-2mm) was 75%±3%, pH was 8.7±0.2, electrical conductivity (salt) EC value (dS / m) was 4.5±0.3, and SAR value (mmol) was... 1 / 2 ·L -1 / 2 ) 19.2±1.3, >0.25mm water-stable aggregates 8.5%±1.2%, soil erosion resistance index 0.42±0.03, available phosphorus / potassium (mg / kg) 5.9±0.4 / 88±4.6.

[0094] (3) Experimental design:

[0095] The randomized block design (2 acres per group, 3 replicates) was used. After planting, irrigation was carried out once a week (in small amounts and frequently to avoid salt stratification), and weeding was done manually. Only control group 2 was sprayed with carbendazim (80g / acre) when black spot disease occurred, while the other groups were not treated. The planted variety was rose "Purple Branch Rose".

[0096] (4) Test results: The test results are shown in Table 4 below.

[0097] Table 4. Multidimensional Effect Verification Analysis of Rose Cultivation Trial

[0098]

[0099] 2.2 Maize Experiment in Sandy Saline-Alkali Land of Wuhai, Inner Mongolia

[0100] (1) Test fertilizers and fertilization methods:

[0101] Processing Group 1:

[0102] Fertilizer: The amount of liquid compound microbial agent added to the bio-fertilizer 1 prepared according to the preparation method in the second set of examples is 0.8%.

[0103] Application method: Use Luyuan commercial organic fertilizer (500 kg / mu) as base fertilizer, mix bio-fertilizer 1 with seeds (0.9 kg / mu) + spray bio-fertilizer 1 at the large trumpet stage (8 kg / mu).

[0104] Processing Group 2:

[0105] Fertilizer: Replace the electrochemical low-hertz water in the preparation method of the second group embodiment with ordinary sterilized water, and the other operations are exactly the same as in the second group embodiment to obtain bio-fertilizer 2;

[0106] Application method: Same as treatment group 1.

[0107] Control group 1:

[0108] Fertilizer: Replace the liquid compound bacterial agent in the preparation method of the second group of examples with a single halophyte Nocardia, and the other operations are exactly the same as in the second group of examples.

[0109] Application method: Same as treatment group 1.

[0110] Control group 2:

[0111] Fertilizers: Sinochem compound fertilizer (15-15-15), Sinochem urea (total nitrogen ≥46%), Sinochem potassium chloride (potassium oxide ≥60%);

[0112] Application method: After evenly spreading Sinochem compound fertilizer (15kg / mu) and Luyuan commercial organic fertilizer (500kg / mu), rotary tillage is carried out. During the large trumpet mouth stage, Sinochem urea (20kg / mu) and Sinochem potassium chloride (5kg / mu) are applied in holes.

[0113] (2) Test location:

[0114] In the maize experimental area of ​​desertified saline-alkali land in Wuhai, Inner Mongolia, the soil is desertified and saline with sand content (0.05-2mm) of 72%±3%, pH of 8.5±0.2, EC value (dS / m) of 3.8±0.3, and soil wind erosion (kg / m²). 2 1.8±0.2.

[0115] (3) Experimental design:

[0116] The randomized block design was used, with each group consisting of 1.5 acres and three replicates. The corn variety "Denghai 618" was planted, and field management was consistent across all groups.

[0117] (4) Test results: The test results are shown in Table 5.

[0118] Table 5. Multidimensional Effect Verification Analysis of Maize Planting Trial

[0119]

[0120] 2.3 Potato Experiment in Sandy and Saline-Alkali Land, Yulin, Shaanxi Province

[0121] (1) Test fertilizers and fertilization methods:

[0122] Processing Group 1:

[0123] Fertilizer: The bio-fertilizer 1 prepared according to the preparation method in the second set of examples has a liquid compound microbial agent addition amount of 6%.

[0124] Application method: Apply Luyuan commercial organic fertilizer (450 kg / mu) as base fertilizer, mix seed with bio-fertilizer 1 (5 kg / mu) + spray bio-fertilizer 1 (6 kg / mu) during seedling stage + spray bio-fertilizer 1 (8 kg / mu) from budding stage to early tuber formation stage.

[0125] Processing Group 2:

[0126] Fertilizer: Replace the electrochemical low-hertz water in the preparation method of the second group embodiment with ordinary sterilized water, and the other operations are exactly the same as in the second group embodiment to obtain bio-fertilizer 2;

[0127] Application method: Same as treatment group 1.

[0128] Control group 1:

[0129] Fertilizer: Replace the liquid compound bacterial agent in the preparation method of the second group of examples with a single halophyte Nocardia, and the other operations are exactly the same as in the second group of examples.

[0130] Application method: Same as treatment group 1.

[0131] Control group 2:

[0132] Fertilizers: Sinochem High Phosphorus Potassium Compound Fertilizer (10-15-20), Luyuan Commercial Organic Fertilizer, Sinochem Compound Fertilizer (18-6-12), and Sinochem High Potassium Compound Fertilizer.

[0133] Application method: Apply Sinochem high phosphorus and potassium compound fertilizer (10-15-20) (35 kg / mu) and Luyuan commercial organic fertilizer (450 kg / mu) as base fertilizer + apply Sinochem compound fertilizer (18-6-12) (15 kg / mu) during the seedling stage + apply Sinochem high potassium compound fertilizer (25 kg / mu) from the budding stage to the early tuber formation stage.

[0134] (2) Test location:

[0135] The potato experimental area in Yulin, Shaanxi Province, is characterized by sandy saline-alkali soil with a sand content (0.05-2mm) of 68%±2%, pH of 8.6±0.2, EC value (dS / m) of 4.1±0.3, and available phosphorus / potassium (mg / kg) of 6.2±0.5 / 90±4.8.

[0136] (3) Experimental design:

[0137] A randomized block design was used, with each group having a planting area of ​​1.5 mu (approximately 0.16 acres) and three replicates. The potato variety "Kexin No. 1" was planted, and field management was consistent across groups.

[0138] (4) Test results: The test results are shown in Table 6.

[0139] Table 6. Multidimensional Effect Verification Analysis of Potato Planting Trial

[0140]

[0141] In summary, the bio-fertilizer prepared by electrochemical low-hertz water in this application has achieved an integrated effect of "sand fixation-salinity reduction-yield increase-quality improvement" on different types of sandy saline-alkali lands in Inner Mongolia and Shaanxi, as well as on roses, corn, and potatoes. It has strong stability and versatility, and is superior to single-strain and conventional fertilizers.

Claims

1. A highly salt-tolerant liquid compound microbial agent suitable for saline-alkali and sandy land, characterized in that, The following functional strains were prepared using electrochemical low-Hertz water through a three-stage sequential inoculation and fermentation process: salt-tolerant restorative bacteria *Nocardia halophyte*, salt-tolerant functional bacteria *Bacillus subtilis* and *Azospirillum brasiliensis*, nitrogen-fixing functional bacteria *Azotobacter chrysogenum*, phosphate-solubilizing functional bacteria *Pseudomonas fluorescens*, potassium-solubilizing functional bacteria *Bacillus mucilaginosus*, biocontrol and growth-promoting bacteria *Trichoderma echinococcus*, and rhizosphere-optimizing bacteria *Trichoderma harzianum*. The inoculation and fermentation preparation includes the following steps: S1: Stress-resistant foundation building period: Add electrolytic low-hertz water to the sterilized main fermenter, and add pre-mixed salt-tolerant repair bacteria and salt-tolerant functional bacteria seed liquid. Adjust the fermentation conditions to the first-order fermentation conditions: pH=7.5±0.3, dissolved oxygen content of 25-30%, temperature of 32℃±0.5℃, humidity of the top space of the fermenter of 90-92%, fermentation time of 23-25 ​​hours; S2: Nutrient Activation Period: After the S1 stage, the conditions are adjusted to the second fermentation time. Pre-mixed seed culture of nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing bacteria is added to the main fermenter. The second fermentation time conditions are: pH=6.8±0.2, dissolved oxygen 30-40%, temperature 32℃±0.5℃, humidity of the top space of the fermenter 91-93%, and fermentation time 17-19 hours. S3: Synergistic Sand Fixation Functional Period: After the fermentation of the S2 stage, the seed liquid of biocontrol and growth-promoting bacteria and rhizosphere optimization bacteria is directly introduced, and salt-tolerant repair bacteria seed liquid is added. Mechanical stirring is turned on and the fermentation conditions are adjusted to the third time sequence: pH=7.0±0.2, dissolved oxygen content is 40-50%, temperature is 32℃±0.5℃, humidity of the top space of the fermenter is 85-88%, and fermentation time is 35-37 hours. When the total viable count in the main fermenter is ≥2.2×10⁻⁶ 10 Fermentation ends when CFU / mL is reached.

2. The highly salt-tolerant microbial liquid compound agent suitable for saline-alkali desertified land as described in claim 1, characterized in that, The volume ratio of the seed liquid of salt-tolerant repair bacteria, salt-tolerant functional bacteria, nitrogen-fixing functional bacteria, phosphorus-solubilizing functional bacteria, potassium-solubilizing functional bacteria, biocontrol and growth-promoting bacteria, and rhizosphere-optimizing bacteria is 3:2:2:1:1:1:

1.

3. The highly salt-tolerant microbial liquid compound agent suitable for saline-alkali desertified land as described in claim 2, characterized in that, The end of stage S1 is marked by: a total viable count ≥ 1 × 10⁻⁶ in the main fermenter. 9 CFU / mL, salt-tolerant remediation bacteria ≥55%; EC value ≤12mS / cm, salinity reduced to 1.5%; contamination rate ≤0.01%; the end of S2 stage is marked by: total viable count ≥1.5×10⁻⁶. 9 The CFU / mL concentration, available phosphorus ≥ 55 mg / L, available potassium ≥ 35 mg / L, nitrogenase activity ≥ 28 nmol C2H4 / (mL·h), system viscosity ≤ 35 cP, and salinity ≤ 1.2% are all indicators of the end of the S3 phase. 10 CFU / mL, with ≥2.2 × 10⁻⁶ bacteria in the viable bacteria. 10 CFU / mL, Trichoderma spores ≥2×10 7 Cells / mL; extracellular polysaccharide ≥12g / L, potassium feldspar decomposition rate ≥30%, oxalic acid content secreted by Trichoderma ≥8mmol / L; Trichoderma spore germination rate ≥92%, Bacillus mucilaginosus silicate enzyme activity ≥15U / mL; fermentation broth salinity ≤1.0%, pH stable at 7.0±0.2; sand grain binding rate ≥65%.

4. The highly salt-tolerant microbial liquid compound agent suitable for saline-alkali desertified land according to claim 2, characterized in that, The preparation and mixing of seed solutions in S1 and S2 include pretreatment, primary seed tank culture, and secondary premix tank proportioning. The specific steps are as follows: Step 1: Pretreatment: Each functional strain was inoculated into its corresponding specialized culture medium and cultured at 30℃ for 24-48 hours until all strains reached the logarithmic growth phase. The salt-tolerant repair bacteria were cultured on LB medium + 0.3% NaCl + 0.02% betaine; the salt-tolerant functional bacteria were cultured on LB medium + 0.3% NaCl + 0.2% proline; the biocontrol and growth-promoting bacteria were cultured on PDA medium + 0.1% arabinose; the nitrogen-fixing functional bacteria were cultured on Assambe medium; the phosphorus-solubilizing functional bacteria were cultured on KB medium + 0.1% calcium phytate + 0.01% MnSO4; the potassium-solubilizing functional bacteria were cultured on basal medium + 2% potassium feldspar powder; and the rhizosphere optimization bacteria were cultured on PDA medium + 0.15% humic acid. All specialized culture media were prepared with electrolytic low-Hertz water and autoclaved at 121℃ for 30 minutes. Step 2: Primary Seed Tank Culture: Configure 8 independent primary seed tanks, and add the strains cultured in Step 1 into different independent primary fermenters for culture and propagation. Step 3: Proportional mixing in secondary premixing tanks: Two premixing tanks were selected based on stress resistance synergy and nutrient dependence to premix the strain seed liquids in two groups. One premixing tank mixed the seed liquids of Haloxylon ammodendron, Bacillus subtilis, and Azotobacter brasiliensis at a volume ratio of 3:1:1, and kept at 32°C and 90-92% humidity at the top of the fermenter for 30 minutes. The other premixing tank mixed the seed liquids of Azotobacter chrysogenum, Pseudomonas fluorescens, and Bacillus mucilage at a volume ratio of 2:1:1, and kept at 32°C and 91-93% humidity at the top of the fermenter for 20 minutes. The premixing tanks were made of 316L stainless steel and equipped with a double-helix agitator, pH / humidity sensor, and sterile feeding port.

5. The highly salt-tolerant microbial liquid compound agent suitable for saline-alkali desertified land according to claim 4, characterized in that, During stage S1, when fermentation reaches the mid-logarithmic growth phase, nutrient factors 0.05% betaine, 10 μM sodium molybdate, and 0.03% proline are added. Two hours after stage S1, during stage S2, nutrient factors 2 g / L food-grade calcium phytate, 5 g / L potassium feldspar powder, and 0.02% 99% pure glutamic acid are added. Four hours after seed culture inoculation in stage S3, nutrient factors 0.2% 70% pure weathered coal-derived humic acid, 0.1% food-grade xanthan gum, and trace element solution are added; the trace element solution includes 0.001% Fe. 2+ and 0.0005%Mn 2+ .

6. The highly salt-tolerant microbial liquid compound agent suitable for saline-alkali desertified land according to claim 5, characterized in that, The method of adding betaine, sodium molybdate and proline in S1 is as follows: first inject betaine through a sterile peristaltic pump with an accuracy of ±0.1 mL / min, and then inject the mixture of sodium molybdate and proline simultaneously after an interval of 30 seconds. The nutrient factor addition method in S2 is as follows: after mixing calcium phytate and glutamic acid, it is injected synchronously through the annular liquid distributor at the bottom of the tank with a 0.5mm aperture. After 20 minutes, potassium feldspar powder and electrolytic low-hertz water are mixed to form a 10% suspension, which is injected through the central tube of the stirring shaft that extends 10cm below the liquid surface. At the same time, the stirring speed is increased from 160r / min to 180r / min. Calcium phytate needs to be added continuously in 3 batches, with an interval of 4 hours between each batch. Glutamic acid and calcium phytate are mixed and injected synchronously through the annular liquid distributor at the bottom of the tank. The method of adding nutrients in S3 is as follows: after mixing the three into a sterile solution, add the solution in the form of atomized liquid through the atomizing nozzle on the top of the tank. The atomization covers ≥90% of the cross section inside the tank, and the stirring speed is increased to 200r / min.

7. A highly salt-tolerant bio-fertilizer suitable for saline-alkali and sandy land, characterized in that, It is prepared using the highly salt-tolerant microbial liquid compound inoculant suitable for saline-alkali desertified land as described in any one of claims 1-6.

8. The high-salt-tolerant bio-fertilizer suitable for saline-alkali and sandy land according to claim 7, characterized in that, The method for preparing the bio-fertilizer includes: A1: The aforementioned microbial liquid compound inoculant, lignite, wheat bran, soybean meal, and electrolytic low-hertz water 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 for the first solid-state fermentation. The solid-state fermentation system automatically adjusts the temperature during the fermentation process through a temperature sensor linked to the intermittent micro-burst oxygen frequency: the initial stage temperature is 42-45℃, maintained for 22-24 hours; the peak stage temperature is 60-65℃, maintained for 46-48 hours; and the final stage temperature is 40-45℃, maintained for 22-24 hours. A2: The material obtained from the first solid-state fermentation in A1 is rearranged into the aging and fermentation production line for a second aging and fermentation through an intelligent material distribution system: the naturally piled material is deeply decomposed by utilizing the residual heat and residual microorganisms of the material, and the fermentation ends after 10-12 days.

9. The high-salt-tolerant bio-fertilizer suitable for saline-alkali and sandy land according to claim 8, characterized in that, The completion of the first solid-state fermentation in stage A1 is marked 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. The completion of the second aging fermentation in stage A2 is marked by the following: the material naturally dehumidifies 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 6.5-7.5, and the number of beneficial live bacteria is ≥200 million / g.

10. The high-salt-tolerant bio-fertilizer suitable for saline-alkali and sandy land according to claim 9, characterized in that, In A1, the total amount of solid components—80-100 mesh 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 initial moisture content of the lignite powder is 10-15%, and the initial moisture content of both wheat bran and soybean meal is 8-12%. After the above solid components are mixed evenly, electrolytic low-hertz water is added and stirred thoroughly. Simultaneously, a 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 mixture reaches 50-60%.

Images