A stress-resistant microbial agent and its application in multi-element complex pollution control

Abstract

The application discloses an anti-reversibility microbial agent and application thereof in multi-element composite pollution prevention and control, and belongs to the technical field of environmental microbiology and soil remediation. The microbial agent is prepared from a modified bran carrier and a composite bacterial liquid of Bacillus dabanchenensis and Bacillus amyloliquefaciens. After application in saline-alkali and heavy metal composite pollution soil, the microbial agent can significantly improve rhizosphere colonization capacity of the functional strains, greatly reduce soil available Cd and Pb contents, improve soil tillage, inhibit heavy metal eluviation and migration, reduce Cd and Pb contents in corn kernels, and significantly improve crop yield, so that the synergistic effect of the two strains, environmental remediation and agricultural safety are realized.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology and soil remediation technology, specifically relating to a stress-resistant microbial agent and its application in the control of multi-component pollution. Background Technology

[0002] In the process of industrialization and agricultural intensification, the complex pollution caused by soil salinization and heavy metal pollution not only affects the normal growth and development of crops, but also threatens human health through the food chain. Therefore, developing remediation technologies suitable for soils with complex salinity and heavy metal pollution is of great practical significance.

[0003] Currently, the main methods for remediating heavy metal pollution in soil include physical remediation, chemical remediation, and bioremediation. Among them, microbial remediation has received widespread attention due to its advantages such as environmental friendliness, low cost, and low risk of secondary pollution. By applying beneficial microorganisms with heavy metal tolerance, adsorption, or passivation functions, the bioavailability of heavy metals in soil can be effectively reduced, thereby decreasing their migration and accumulation in crops.

[0004] However, under combined saline-alkali and heavy metal stress, the survival, colonization, and activity of functional microorganisms are often significantly inhibited, leading to unstable remediation effects. The selection and modification of carrier materials play a crucial role in protecting functional strains, prolonging their survival time, and enhancing their competitiveness in soil. Agricultural byproducts such as wheat bran are often used as microbial carriers due to their high organic matter content, large specific surface area, and low price. However, untreated wheat bran contains high levels of anti-nutritional factors such as phytic acid, and its pore structure is not ideal, affecting its adsorption and protective effects on microorganisms.

[0005] Furthermore, current technologies mostly employ single-functional strains for soil remediation, which struggle to address the dual pressures of saline-alkali stress and heavy metal pollution simultaneously. The potential for synergistic effects of multifunctional strains has not been fully explored, particularly the combined application of strains with multiple functions such as salt tolerance, extracellular polymer production, and promotion of heavy metal passivation remains relatively limited.

[0006] Therefore, developing a stress-resistant microbial agent suitable for soils contaminated with saline-alkali and heavy metals, possessing excellent carrier performance and synergistic effects of composite strains, has significant application prospects and industrialization value. Summary of the Invention

[0007] In view of the above-mentioned prior art, the purpose of this invention is to provide a stress-resistant microbial agent and its application in the control of multi-component complex pollution.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a stress-resistant microbial agent, which is prepared from a modified bran carrier and a composite bacterial solution; The compound bacterial solution consists of Halobacillus dabanensis NBL-BS214 culture medium and Bacillus amyloliquefaciens NBL-B11002 culture medium.

[0009] The *Haloxylon ammodendron* strain NBL-BS214 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20221370. This strain has been described in the published patent CN 116286461 B.

[0010] The Bacillus amyloliquefaciens NBL-B11002 strain is deposited at the China Center for Type Culture Collection (CCTCCNO: M 20232184) and has been described in the published patent CN 117660243B.

[0011] It should be noted that although there are existing reports on the use of Bacillus amyloliquefaciens for heavy metal adsorption or passivation, these are mostly conducted under conditions of single heavy metal pollution or non-salt-alkali stress in heavy metal-contaminated soil. When this invention is applied to a more severe environment of saline-alkali and heavy metal compound pollution (pH ≥ 8.0, total salt ≥ 0.3%), the colonization and passivation effects of Bacillus amyloliquefaciens alone decrease significantly due to salt-alkali stress. One of the ingenious aspects of this invention is the introduction of the highly salt-alkali-tolerant Bacillus tasaka (which maintains high activity even in extreme saline-alkali environments) in combination with it. The superior survival and colonization ability of the former in compound-contaminated soil provides a suitable microenvironment for the latter, thus enabling a synergistic effect under compound stress.

[0012] Preferably, the method for preparing the modified bran carrier includes the following steps: (1) Soak wheat bran in a 0.3%-0.8% hydrochloric acid solution at 50-70℃ for 20-40 min; (2) After acid soaking, wash and drain until the moisture content is 55%-65%; (3) The moistened slag is subjected to steam explosion treatment at 140-160℃ and 0.50-0.70 MPa for 10-20 minutes; (4) Dry and pulverize to obtain the modified bran carrier.

[0013] The combined modification process of "acid impregnation + steam explosion" employed in this application can more efficiently remove phytic acid (an anti-nutritional factor) and significantly improve the specific surface area, porosity, and water-holding capacity of the carrier. Experiments show that after modification, the phytic acid content is reduced to 38.8% of that in unmodified bran, while the soluble dietary fiber content is increased to 9.5%, providing a nutritionally and protectively optimized microenvironment for the attachment and proliferation of functional strains under stress.

[0014] Preferably, the effective viable count of Halobacillus dabanensis NBL-BS214 culture medium is ≥1.0 × 10⁻⁶. 9 The effective viable count of Bacillus amyloliquefaciens NBL-B11002 culture medium is ≥1.0 × 10⁻¹⁰ CFU / mL. 9 CFU / mL.

[0015] Preferably, the ratio of the effective viable counts of *Hydrocetamol* tasaka to *Bacillus amyloliquefaciens* in the compound bacterial solution is (0.5-2):(0.5-2). More preferably, it is 1:1.

[0016] Preferably, the mass ratio of the modified bran carrier to the compound bacterial solution is (3-7):1. More preferably, it is 5:1.

[0017] The method for preparing the stress-resistant microbial agent includes the following steps: (1) Preparation of modified bran carrier; (2) Culture Bacillus tasakae NBL-BS214 and Bacillus amyloliquefaciens NBL-B11002 separately until the effective viable count is ≥1.0×10⁻⁶. 9 CFU / mL, mixed at a volume ratio of (0.5-2):(0.5-2) to obtain a compound bacterial solution; (3) Mix the modified bran carrier and the compound bacterial solution at a mass ratio of (3-7):1 to obtain wet material, dry it in a ventilated environment at 35-45℃, and then crush it to obtain the stress-resistant microbial agent.

[0018] The preparation method of this invention only involves conventional carrier modification, liquid fermentation culture, mixed adsorption and low temperature drying. The process steps are simple and easy to scale up. Moreover, the raw materials are only agricultural by-product wheat bran and two strains of functional bacteria, which are low in cost and environmentally friendly.

[0019] In a second aspect, the present invention provides the use of the above-mentioned stress-resistant microbial agent in any of the following (a)-(e): (a) Reduce the content of available heavy metals in soils contaminated with saline-alkali soil and heavy metals; (b) Inhibit the leaching and migration of heavy metals in soils contaminated with both saline-alkali and heavy metals; (c) Reduce the heavy metal content in crop grains grown in soils with combined saline-alkali and heavy metal pollution; (d) Improve the tilth of soils contaminated with saline-alkali soil and heavy metals; (e) Increase crop yields in soils contaminated with saline-alkali and heavy metals.

[0020] The soil contaminated with saline-alkali and heavy metals has a pH ≥ 8.0, total salt content ≥ 0.3%, total Cd content ≥ 0.5 mg / kg, and total Pb content ≥ 100 mg / kg.

[0021] Preferably, the heavy metal is Cd and / or Pb.

[0022] From the perspective of the application effect of microbial agents, this application achieves the dual goals of effectively inhibiting the dynamic leaching and migration of heavy metals in soils with combined saline-alkali and heavy metal pollution and significantly reducing the heavy metal content in crop grains. Its mechanism of action is different from the passive remediation methods in existing technologies that may rely on leaching and migration to remove heavy metals from the topsoil.

[0023] In conventional soil heavy metal remediation practices, when materials (such as clay minerals, biochar, and unmodified organic materials) are applied to the soil, their fixation of heavy metals mainly relies on physical adsorption or simple ion exchange. Under natural rainfall or irrigation conditions, water infiltration has two consequences: (1) Physical scouring: Water flow washes away soil particles and the heavy metals adsorbed on their surface, carrying them to deeper soil layers. Especially when the soil structure is poor (such as high bulk density, low porosity, and few large-diameter water-stable aggregates), water flow tends to form a preferential flow, accelerating the vertical migration of heavy metals.

[0024] (2) Desorption and release: The adsorption of heavy metals by some materials is reversible. With the extension of leaching time and changes in pH or ionic strength of the leaching solution, the heavy metals that were originally "fixed" may be re-desorbed into the liquid phase and seep down with the water.

[0025] Under the aforementioned traditional model, the reduction in the content of available heavy metals in the soil may seem like a successful restoration in the short term, but in reality, the heavy metals are only transferred from the surface to the deeper layers and are not truly removed from the soil ecosystem.

[0026] This application breaks away from the technical bias that "inhibiting leaching inevitably leads to increased grain content" or "reducing heavy metals in grains must first be achieved through leaching." Specifically, the modified bran carrier, rich in soluble dietary fiber, significantly reduces bulk density, increases the content of >0.25 mm water-stable aggregates and total porosity after being applied to the soil, improving soil structure and thus reducing preferential flow and physical erosion of heavy metals by water. Simultaneously, the complex bacterial strains (especially Bacillus amyloliquefaciens) convert available Cd and Pb into insoluble or poorly migratable forms through extracellular polymer complexation and microbial-induced mineralization, significantly reducing the risk of heavy metal desorption in the liquid phase. The synergistic effect of these two factors leads to a substantial reduction in the cumulative leaching loss of Cd and Pb.

[0027] On the other hand, *Halophilic bacillus tasaka* exhibits extremely strong salt and alkali tolerance, and can still colonize extensively in the rhizosphere of maize even under stress environments with pH ≥ 8.0 and total salt ≥ 0.3%. When heavy metal ions in the soil solution migrate towards the roots, they are first adsorbed, complexed, or passivated by the high-density bacterial cells in the rhizosphere, thus significantly reducing their contact with the roots. Simultaneously, *Bacillus amyloliquefaciens* continuously converts readily available heavy metals in the rhizosphere into stable forms, maintaining absorbable heavy metal levels in the rhizosphere microdomain at extremely low levels. Therefore, even under conditions where leaching is strongly inhibited and heavy metals are mainly retained in the topsoil, the Cd and Pb contents in maize kernels can still be reduced.

[0028] The beneficial effects of this invention are as follows: 1. This invention is the first to specifically combine a salt-tolerant bacterial strain (Haloxylon ammodendron) with a heavy metal passivating bacterial strain (Bacillus amyloliquefaciens), along with a functional carrier, successfully overcoming the technical bottleneck of the functional bacteria's inability to survive and colonize under dual stress of salt, alkali, and heavy metals. The two strains complement each other and synergistically enhance each other under adverse conditions, achieving a remediation effect of "1+1>2". Example data show that the passivation rate of the compound bacterial agent on available Cd and Pb in soil is significantly better than that of a single strain.

[0029] 2. This invention verified the inhibitory effect of the prepared microbial agent on the vertical migration of heavy metals through field leaching disc experiments. The compound microbial agent reduced the cumulative leaching of Cd and Pb by 83.2% and 79.4%, respectively (compared to the blank control group). Furthermore, while remediating heavy metal pollution, the microbial agent of this invention can improve the physical structure of the soil (reducing bulk density, increasing porosity and aggregates), enhance soil tilth, and ultimately increase crop yield (28.9% increase in maize yield) and reduce the heavy metal content in grains (69.2% reduction in Cd and 56.2% reduction in Pb), achieving a win-win situation for environmental remediation and agricultural production safety.

[0030] 3. The core raw materials of this invention are only wheat bran, an agricultural by-product, and two strains of environmental functional bacteria. They can be obtained through a simple process of "acid-explosive modification + mixed drying". No expensive materials or complex equipment are required, and it has good prospects for industrial application. Detailed Implementation

[0031] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0032] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0033] Example 1: Stress-resistant microbial agents 1. Preparation of modified bran carrier (1) Take 1000g of wheat bran, add 3000g of 0.5% hydrochloric acid solution, soak in a 60℃ water bath for 30min, stirring 3 times during the process.

[0034] (2) After the acid soaking is completed, filter out the hydrochloric acid solution, wash the skin residue three times with deionized water, and drain until the skin residue has a moisture content of about 55%-65% (it can be clumped when squeezed and easily dispersed when touched).

[0035] (3) The above-mentioned wet slag was subjected to steam explosion. The steam explosion treatment conditions were: 150℃, 0.60 MPa for 15 min.

[0036] (4) Collect the material after steaming and explosion treatment, dry it with hot air at 60℃ until the moisture content is ≤8%, and crush it through a 60-mesh sieve to obtain the modified bran carrier.

[0037] 2. Preparation of compound bacterial solution (1) Bacillus dasaka halophilus NBL-BS214 and Bacillus amyloliquefaciens NBL-B11002 were inoculated onto LB solid plate medium and activated in a constant temperature incubator at 35℃ for 36h.

[0038] (2) Pick activated single colonies and inoculate them into 250 mL Erlenmeyer flasks containing 100 mL LB liquid medium. Incubate at 35 °C and 180 rpm with shaking for 18 h until the bacterial culture OD reaches its maximum. 600 Reaching a value of 1.0 yields a first-grade seed solution.

[0039] (3) The primary seed culture was transferred at an inoculation rate of 5% (v / v) to 1000 mL Erlenmeyer flasks containing 500 mL of LB liquid medium. Both strains were cultured with shaking at 35℃ and 180 rpm until the effective viable counts of the secondary seed culture of Bacillus tasakae NBL-BS214 and Bacillus amyloliquefaciens NBL-B11002 reached 1.0 × 10⁻⁶. 9 Stop when CFU / mL.

[0040] (4) Mix the secondary seed liquids of the two strains in a sterile mixing tank at a volume ratio of 1:1 to obtain a compound bacterial solution.

[0041] 3. Preparation of stress-resistant microbial agents (1) The modified bran carrier and the compound bacterial solution were placed in a mixer at a mass ratio of 5:1, stirred at 30 rpm for 20 min at room temperature, and then left to stand for 30 min to obtain the wet material after adsorption loading.

[0042] (2) The wet material after adsorption and loading is evenly spread in a stainless steel tray and dried in a ventilated environment at 42°C for 6 hours. The dried material is then crushed by a pulverizer to obtain a loose powdery stress-resistant microbial agent.

[0043] Comparative Example 1: The difference from Example 1 is that unmodified wheat bran was used as a carrier in the preparation of the stress-resistant microbial agent.

[0044] Comparative Example 2: The difference from Example 1 is that only Bacillus tasakae NBL-BS214 secondary seed culture (effective viable count 1.0 × 10⁻⁶) was added during the preparation of the stress-resistant microbial agent. 9 (CFU / mL).

[0045] Comparative Example 3: The difference from Example 1 is that only Bacillus amyloliquefaciens NBL-B11002 secondary seed culture (effective viable count 1.0 × 10⁻⁶) was added during the preparation of the stress-resistant microbial agent. 9 (CFU / mL).

[0046] Example 2: Characteristics of the modified carrier and its effect on the colonization ability of functional strains in mixed-contaminated soil The tested soil samples were collected from the 0–20 cm topsoil layer of a saline-alkali farmland in Shandong Province. After natural air drying, the samples were sieved through a 2 mm nylon sieve for later use. Soil physicochemical properties: pH 8.2 (alkaline / saline-alkali soil), total salt content 0.32% (moderate salinization), total Cd content 0.96 mg / kg, total Pb content 158 ​​mg / kg, organic matter content 12.5 g / kg, cation exchange capacity 10.8 cmol / kg. This soil exhibits characteristics of both saline-alkali stress and heavy metal pollution.

[0047] 1. Physicochemical properties of carrier materials The physicochemical properties of the carriers used in Example 1 and Comparative Example 1 are shown in Table 1.

[0048] Table 1: Physicochemical properties of different carrier materials Phytic acid is the main anti-nutritional factor in wheat bran that inhibits heavy metal adsorption and microbial colonization. As shown in Table 1, the phytic acid content of the modified wheat bran used in Example 1 was reduced to 1.52%, which is only 38.8% of that of the unmodified wheat bran. In addition, the specific surface area, porosity, soluble dietary fiber content and water holding capacity of the modified carrier were significantly improved, providing a better microenvironment for functional strains.

[0049] 2. Potted plant experiment A pot experiment was conducted, with each pot containing 2.0 kg of test soil. Three treatment groups were set up: a blank control group, Example 1 group, and Comparative Example 1 group, with each group replicated three times. Among them: Blank control group: No bacterial agent was applied; Example 1 group: The microbial agent prepared in Example 1 group was applied at 0.5% of the soil weight; Comparative Example 1: The microbial agent prepared in Comparative Example 1 was applied at 0.5% of the soil weight.

[0050] Thoroughly mix the corresponding inoculant for each treatment with the soil in the pots. Sow 3 corn seeds (variety: Ludan 9088) in each pot, and after emergence, transplant 2 seedlings per pot. The growth cycle is 90 days. Water and fertilize according to local conventional field management methods for each treatment, and maintain consistent management practices.

[0051] Destructive sampling was performed at the maize maturity stage (90 days), collecting samples from the roots, stems and leaves, and kernels of the maize plant. All soil in the pots was poured out, and the compacted soil attached to the maize roots was carefully collected as rhizosphere soil. Using the plate count method, the rhizosphere soil samples were serially diluted and spread onto selective media containing 5.0 g / L peptone, 1.0 g / L yeast extract, 100.0 g / L NaCl, and 15.0 g / L agar, respectively. After incubation at 35℃ for 48 h, single colonies were picked and identified morphologically and molecularly. The colony counts of *Bacillus tasakae* and *Bacillus amyloliquefaciens* were counted, and the viable bacteria count in the rhizosphere soil was calculated. The results are shown in Table 2.

[0052] Table 2: Effects of different vectors on bacterial colonization (90-day pot experiment) Table 2 shows that the target strain was not detected in the blank control group, indicating no exogenous bacterial agent contamination. Compared with the unmodified bran carrier, the modified bran carrier of this invention increased the number of viable colonies of *Bacillus tasakae* and *Bacillus amyloliquefaciens* in the rhizosphere of maize by 2.79 times and 2.86 times, respectively (P<0.05), demonstrating that the modified carrier effectively protects the survival and colonization of functional strains under saline-alkali-heavy metal combined stress.

[0053] Example 3: Evaluation of the effect of stress-resistant microbial agents on heavy metal retention and leaching inhibition 1. Experimental Methods Using the same test soil as in Example 2, maize (variety: Ludan 9088) was selected as the indicator crop for field experiments.

[0054] A randomized block design was used, with 4 treatments, each with 3 replicates, and a cell size of 20 m². 2 (4 m × 5 m), a 1 m wide protective row is set between each section. Specific details are as follows: Table 3: Grouping Processing Before sowing, the land was rotary tilled and prepared. The inoculants for each treatment were mixed with an appropriate amount of fine soil and evenly spread on the corresponding plots, then rotary tilled to incorporate them into the 0-20 cm topsoil layer. Corn was sown using the hill-sowing method, with a row spacing of 60 cm and a plant spacing of 25 cm. During the growing season, routine water and fertilizer management was applied (basal application of compound fertilizer N-P2O5-K2O=15-15-15, 40 kg / mu; topdressing with urea at the large trumpet stage), consistent with all treatments.

[0055] At the corn harvest period (90 days after sowing), soil samples were collected from the 0-20 cm topsoil layer in each plot using the "S"-shaped five-point sampling method. After mixing, 1 kg of the samples were taken, air-dried, and sieved for experimental testing.

[0056] (1) Determination of the passivation effect of heavy metals in soil Accurately weigh 4.00 g (±0.01 g) of sieved soil sample into a 50 mL centrifuge tube, and add 40 mL of 0.01 mol / L CaCl2 solution (soil-to-water ratio 1:5). Seal tightly and incubate at 25℃ with shaking (200 r / min) for 2 h. Then centrifuge at 4000 r / min for 10 min, and filter the supernatant through a 0.45 μm aqueous filter membrane. The content of available Cd or Pb in the filtrate was determined by atomic absorption spectrophotometry.

[0057] Using the blank control group (without bacterial agent) as a control, the passivation rate was calculated using the following formula: Passivation rate (%) = (1-C) 处理 ÷C 对照 ) × 100%, where: C 处理 Soil available Cd or Pb content (mg / kg) in treatment groups (Comparative Example 2, Comparative Example 3, Example 1); C 对照 : Content of available Cd or Pb in soil of blank control group (mg / kg).

[0058] The test results are shown in Table 4.

[0059] Table 4: Effects of different treatments on soil available Cd and Pb content (90 days) Table 4 shows that, compared with the blank control group, all bacterial treatments significantly reduced the content of available Cd and Pb in the soil (P<0.05). Specifically, the passivation rates of Comparative Example 2 (single-strain *Bacillus tasaka*) for Cd and Pb were 30.8% and 28.7%, respectively; while the passivation rates of Comparative Example 3 (single-strain *Bacillus amyloliquefaciens*) were 35.9% and 35.7%, respectively. The passivation effects of the two single strains had different emphases but were generally similar.

[0060] The passivation rates of Cd and Pb in Example 1 (dual-strain compound) reached 76.9% and 74.9%, respectively, which were significantly higher than those of any single bacterial agent group (P<0.01), showing a typical "1+1>2" synergistic effect and significantly improving the passivation efficiency of heavy metals in compound contaminated soil.

[0061] (2) Effect of heavy metal leaching control (field leaching tray method) A leaching tray (30 cm in diameter, 30 cm deep, made of PVC, with a funnel-shaped bottom connected to a polyethylene collection bottle, pre-buried in the pit below, with a sealed cap to prevent evaporation and contamination) was buried in the center of each experimental plot. The tray was ensured to be in close contact with the bottom of the topsoil, and the original soil was backfilled on top. During the four key growth stages of maize—after sowing, jointing, tasseling, and harvest—within 24 hours after each natural rainfall or artificial irrigation, the collection bottle was retrieved to record the leaching volume. 50 mL of the leaching solution was collected in a polyethylene bottle and immediately brought back to the laboratory. The leaching solution was filtered through a 0.45 μm filter membrane, and the Cd and Pb concentrations were determined using inductively coupled plasma mass spectrometry (ICP-MS). Leaching loss calculation: Single urination loss (mg / m³) 2 = Leachate concentration (mg / L) × Leachate volume (L) / Leachate tray area (m²) 2 ) Cumulative leukorrhea = Sum of single leukorrhea losses at each reproductive stage The test results are shown in Table 5.

[0062] Table 5: Effects of different treatments on Cd and Pb leaching and migration (cumulative over the entire growth period) Table 5 shows that all microbial agents effectively reduced the leaching and migration of Cd and Pb in the soil. Compared with the blank control group, Comparative Group 2 (single-strain Halophytum tasaka) reduced the cumulative leaching of Cd and Pb by 38.8% and 33.3%, respectively; Comparative Group 3 (single-strain Bacillus amyloliquefaciens) reduced them by 43.4% and 39.2%, respectively. The leaching control effects of the two single strains were similar.

[0063] In Example 1 (dual-strain combination), the cumulative leaching amounts of Cd and Pb were 0.48 mg / m³. 2 and 2.1 mg / m 2 Compared to the blank control, the levels were reduced by 83.2% and 79.4%. In terms of absolute reduction, the reduction in Cd in Example 1 group was 2.38 mg / m³. 2 (2.86 - 0.48), which is greater than the sum of the reductions in comparative groups 2 and 3 (1.11 + 1.24 = 2.35 mg / m²). 2 The reduction in Pb was 8.1 mg / m³. 2 (10.2 - 2.1), which is also greater than the sum of the reductions in the two individual groups (3.4 + 4.0 = 7.4 mg / m²). 2This indicates that the combined treatment with two strains exhibits a significant synergistic effect in controlling the leaching and migration of heavy metals. The reason for this may be that *Haloxylon ammodendron* provides a suitable microenvironment for *Bacillus amyloliquefaciens* under saline-alkali stress, and both strains jointly promote the adsorption, complexation, and fixation of heavy metals, thereby more effectively reducing the risk of downward migration of heavy metals.

[0064] Example 4: Evaluation of the impact of stress-resistant microbial agents on soil tilth. Based on the experiment in Example 3, soil samples from the 0–20 cm topsoil layer were collected from each plot at the corn harvest period (90 days after sowing) using an “S”-shaped five-point sampling method. The samples were mixed thoroughly, and 1 kg was taken, air-dried, and sieved. The following indicators were measured: (1) Soil bulk density: ring sampler method (volume 100 cm³) 3 The ring cutter is vertically pressed into the soil surface, removed, and excess soil is discarded. The soil is then dried at 105℃ until a constant weight is achieved, and weighed. Bulk density (g / cm³) = Dry soil weight (g) / Ring cutter volume (cm³) 3 ).

[0065] (2) Total porosity: Calculation formula: Total porosity (%) = (1 - bulk density / soil specific gravity) × 100%, soil specific gravity is 2.65 g / cm³ 3 count.

[0066] (3) Content of water-stable aggregates >0.25 mm: wet sieving method. Take 50 g of air-dried soil sample (passing through a 2 mm sieve), place it on the top of a sieve (0.25 mm aperture), and shake it vertically in water at 30 times / min for 30 min. Collect the residual aggregates on the sieve, dry them, weigh them, and calculate the percentage of the total mass of the soil sample.

[0067] (4) Permeability coefficient: An infiltration ring (20 cm in diameter and 25 cm in height) is embedded in the plot, and water is injected to maintain a constant water head (5 cm). The amount of water seeping per unit time is recorded, and the permeability coefficient (cm / s) is calculated.

[0068] (5) Plant height and ear height: At maturity, 10 corn plants were randomly selected from each plot, and the height from the ground to the top of the tassel (plant height) and the height from the ground to the node where the female ear grows (ear height) were measured with a tape measure.

[0069] (6) Plot yield: Harvest all ears of grain from each plot, air dry and thresh them, weigh them, calculate the yield based on the standard moisture content (14%), and then convert it to yield per mu (666.7 m). 2 ).

[0070] (7) Heavy metal content in grains: Take grain samples, wash with deionized water, dry at 80℃ to constant weight, and pulverize. Weigh 0.5 g into a digestion vessel, add 10 mL of a nitric acid-perchloric acid (4:1, v / v) mixture, digest at 180℃ until the solution is clear, remove the acid, and bring the volume to 25 mL. Determine the Cd and Pb contents using ICP-MS. Conduct a safety evaluation according to GB 2762-2022 "National Food Safety Standard - Limits of Contaminants in Food".

[0071] The test results are shown in Tables 6 and 7.

[0072] Table 6: Effects of different treatments on soil tillage Table 6 shows that, compared with the blank control group, all bacterial treatments improved the soil physical structure to some extent. Specifically, Comparative Example 2 (single *Bacillus tasakae*) and Comparative Example 3 (single *Bacillus amyloliquefaciens*) reduced soil bulk density by 2.1% and 2.8%, respectively; increased total porosity by 1.1 and 1.5 percentage points, respectively; increased the content of >0.25 mm water-stable aggregates by 7.6% and 11.9%, respectively; and increased permeability by 6.3% and 9.0%, respectively. The improvement effects of the two single bacterial strains were similar.

[0073] The improvement effect of the first group (two strains combined) was the most significant: compared with the blank control group, the soil bulk density decreased by 7.1% (to 1.31 g / cm³). 3 The total porosity increased by 3.8 percentage points (to 50.6%), the content of water-stable aggregates >0.25 mm increased by 26.2% (to 38.1%), and the permeability increased by 21.3% (to 3.25 × 10⁻⁶). -5 (cm / s). Compared with the two single-strain groups, the Example 1 group showed significant advantages in all indicators, indicating that the combination of dual strains and modified carrier can synergistically improve the tilth of saline-alkali-heavy metal contaminated soil, which is beneficial to crop root growth and water-air coordination.

[0074] Table 7: Effects of different treatments on maize growth, yield, and heavy metal content in grains. Table 7 shows that the application of microbial agents significantly promoted maize growth, increased yield, and reduced heavy metal content in grains. Compared with the blank control group, the plant height of control groups 2 and 3 increased by 6.0% and 8.8%, respectively; the ear height increased by 7.4% and 11.6%, respectively; and the yield per mu increased by 10.9% and 15.1%, respectively. The Cd content in the grains decreased by 26.9% and 38.5%, respectively, and the Pb content decreased by 25.0% and 28.1%, respectively. Single-strain treatment has shown good pollution reduction and yield-increasing effects.

[0075] Example 1 (dual-strain compound) showed the best performance: plant height (252 cm) increased by 17.2% compared to the control, ear height (115 cm) increased by 21.1%, and yield per mu (625 kg) increased by 28.9%. Most importantly, the Cd content (0.08 mg / kg) and Pb content (0.14 mg / kg) in the kernels of Example 1 were reduced by 69.2% and 56.3% respectively compared to the control, and significantly lower than the two single-strain groups. According to GB 2762-2022 "National Food Safety Standard - Limits of Contaminants in Food", the limit for Cd in corn is 0.1 mg / kg. The Cd content in the kernels of Example 1 (0.08 mg / kg) met the national standard, while the control (0.26 mg / kg) and the comparative group (0.16~0.19 mg / kg) exceeded the limit. This indicates that the microbial agent of this invention has outstanding advantages in ensuring the safety of agricultural products.

[0076] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A stress-resistant microbial agent, characterized in that, The stress-resistant microbial agent is prepared from a modified wheat bran carrier and a compound bacterial solution; The compound bacterial solution consists of Halobacillus dabanensis NBL-BS214 culture medium and Bacillus amyloliquefaciens NBL-B11002 culture medium.

2. The stress-resistant microbial agent according to claim 1, characterized in that, The *Haloxylon ammodendron* NBL-BS214 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20221370; the *Bacillus amyloliquefaciens* NBL-B11002 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20232184.

3. The stress-resistant microbial agent according to claim 1, characterized in that, The preparation method of the modified bran carrier includes the following steps: (1) Soak wheat bran in a 0.3%-0.8% hydrochloric acid solution at 50-70℃ for 20-40 min; (2) After acid soaking, wash and drain until the moisture content is 55%-65%; (3) The moistened slag is subjected to steam explosion treatment at 140-160℃ and 0.50-0.70 MPa for 10-20 min; (4) Dry and pulverize to obtain the modified bran carrier.

4. The stress-resistant microbial agent according to claim 1, characterized in that, The effective viable count of Halobacillus dabanensis NBL-BS214 culture medium is ≥1.0×10⁻⁶. 9 The effective viable count of Bacillus amyloliquefaciens NBL-B11002 culture medium is ≥1.0 × 10⁻¹⁰ CFU / mL. 9 CFU / mL.

5. The stress-resistant microbial agent according to claim 1, characterized in that, In the compound bacterial solution, the effective viable number ratio of Bacillus tasakae to Bacillus amyloliquefaciens is (0.5-2):(0.5-2).

6. The stress-resistant microbial agent according to claim 1, characterized in that, The mass ratio of the modified bran carrier to the compound bacterial solution is (3-7):

1.

7. The stress-resistant microbial agent according to claim 1, characterized in that, The method for preparing the stress-resistant microbial agent includes the following steps: (1) Preparation of modified bran carrier; (2) Culture Bacillus tasakae NBL-BS214 and Bacillus amyloliquefaciens NBL-B11002 separately until the effective viable count is ≥1.0×10⁻⁶. 9 CFU / mL, mixed at a volume ratio of (0.5-2):(0.5-2) to obtain a compound bacterial solution; (3) Mix the modified bran carrier and the compound bacterial solution at a mass ratio of (3-7):1 to obtain wet material, dry it in a ventilated environment at 35-45℃, and then crush it to obtain the stress-resistant microbial agent.

8. The use of the stress-resistant microbial agent according to claim 1 in any one of the following (a)-(e): (a) Reduce the content of available heavy metals in soils contaminated with saline-alkali soil and heavy metals; (b) Inhibit the leaching and migration of heavy metals in soils contaminated with both saline-alkali and heavy metals; (c) Reduce the heavy metal content in crop grains grown in soils with combined saline-alkali and heavy metal pollution; (d) Improve the tilth of soils contaminated with saline-alkali soil and heavy metals; (e) Increase crop yields in soils contaminated with saline-alkali and heavy metals.

9. The application according to claim 8, characterized in that, The soil contaminated with saline-alkali and heavy metals has a pH ≥ 8.0, total salt content ≥ 0.3%, total Cd content ≥ 0.5 mg / kg, and total Pb content ≥ 100 mg / kg.

10. The application according to claim 8, characterized in that, The heavy metal is Cd and / or Pb.

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