Application of compound microbial inoculants and their combination with mulberry and silkworm excrement organic fertilizer in the remediation of thallium-contaminated soil

Abstract

This invention relates to the field of compound microbial agents, particularly to the application of compound microbial agents and their combination with mulberry and silkworm excrement organic fertilizer in the remediation of thallium-contaminated soil. This invention uses a compound microbial agent prepared by combining *Bacillus megaterium* with *Bacillus subtilis* SEM-2 and *Bacillus cereus* SEM-15 derived from silkworm excrement. This compound microbial agent synergistically promotes the growth of mulberry trees and the adsorption and transfer of the heavy metal thallium, thereby improving the remediation efficiency of thallium in soil. Furthermore, the method provided by this invention, through the combination of silkworm excrement organic fertilizer, compound microbial agent, and mulberry trees, further promotes the growth of mulberry trees, accelerates the transfer and accumulation of the heavy metal thallium, and fully leverages the synergistic effect of 1+1+1>3. This is a combined remediation method that can synergistically improve thallium remediation efficiency, regulate the distribution of thallium within mulberry trees, and ensure the safety of mulberry leaf feed, possessing significant practical significance and application value.

Description

Technical Field

[0001] This invention relates to the field of compound microbial agents, and in particular to the application of compound microbial agents and their combination with mulberry and silkworm excrement organic fertilizer in the remediation of thallium-contaminated soil. Background Technology

[0002] Thallium (Tl) is a highly toxic heavy metal, far more toxic to mammals than conventional heavy metals such as cadmium and lead. In pyrite mining areas, the associated presence of sulfide minerals often leads to severe thallium pollution in the soil and water. Thallium exhibits extremely high mobility and bioaccumulation; therefore, how to economically and effectively remediate soils contaminated with low to medium concentrations of thallium and prevent thallium from entering the human body through the food chain has become an urgent issue in the environmental management of mining areas.

[0003] Currently, remediation technologies for thallium pollution mainly include chemical passivation, soil leaching, and phytoremediation. However, thallium often exists in soil as a by-product, and chemical passivation alone is easily affected by soil acidification and becomes ineffective; physicochemical remediation is costly and easily damages soil structure. While phytoremediation is environmentally friendly, traditional hyperaccumulating plants often have small biomass and slow growth, and the disposal of remediated plants remains unresolved. In recent years, the "remediation while production" model, utilizing economic crops such as mulberry trees to remediate heavy metal-contaminated soil, has attracted attention. However, existing technologies mostly focus on conventional heavy metals such as copper and cadmium, with very little research on the migration, transformation, and accumulation control of thallium in mulberry trees. In particular, under thallium pollution stress, how to improve the extraction efficiency (i.e., transfer efficiency) of thallium from mulberry trees while ensuring the safety of its byproducts (mulberry leaves) as feed is a key bottleneck restricting the practical application of this technology. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a compound microbial inoculant and its application in combination with mulberry and silkworm excrement organic fertilizer for the remediation of thallium-contaminated soil. The compound microbial inoculant provided by this invention can synergistically promote the growth of mulberry trees and the adsorption and transfer of the heavy metal thallium, thereby improving the remediation efficiency of thallium in soil.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a compound microbial agent, characterized in that the active ingredient includes Bacillus subtilis (…). Bacillus sp.) SEM-2, Bacillus cereus ( Bacillus cereus SEM-15 and Bacillus megaterium ( Bacillus megatherium The Bacillus subtilis SEM-2 has a GDMCC No. 60039; the Bacillus cereus SEM-15 has a GDMCC No. 61562; and the Bacillus megaterium has a GDMCC No. 60167.

[0006] Preferably, the ratio of viable bacteria of Bacillus subtilis SEM-2, Bacillus cereus SEM-15 and Bacillus megaterium is 1:1:1.

[0007] Preferably, the compound microbial agent has a live bacteria concentration ≥1×10⁻⁶. 9 Solid bacterial powder with CFU / g, or a live bacteria concentration ≥1×10⁻⁶ 9 Liquid bacterial agent at CFU / mL.

[0008] This invention provides the application of the composite microbial agent described in the above technical solution in one or more of the following: 1) Improve the survival rate of plants in thallium-contaminated soil; 2) Reduce the content of the heavy metal thallium in the soil; 3) Increase the activity of rhizosphere soil enzymes in thallium-contaminated soil; 4) Decompose inorganic phosphorus; 5) Promotes plant growth; 6) Reduce the content of the heavy metal thallium in plant leaves.

[0009] Preferably, the rhizosphere soil enzymes include one or more of acid phosphatase, urease, and catalase.

[0010] Preferably, the plant includes the mulberry tree.

[0011] This invention provides a method for promoting plant growth and remediating thallium-contaminated soil, comprising the following steps: Mulberry trees were planted in thallium-contaminated soil, and the compound microbial agent described in the above technical solution was applied.

[0012] Preferably, the application concentration of the compound microbial agent is ≥10. 6 CFU / mL; the application method includes root irrigation, which is performed once every 25-35 days, with 300-800 mL per plant each time.

[0013] Preferably, the method further includes: applying silkworm excrement organic fertilizer; the application rate of the silkworm excrement organic fertilizer is 0.5-2 tons / mu.

[0014] Preferably, the mulberry tree is Yue Sang No. 11.

[0015] Beneficial effects: This invention provides a compound bacterial agent obtained by combining Bacillus megaterium with Bacillus subtilis SEM-2 and Bacillus cereus SEM-15 derived from silkworm excrement. This compound bacterial agent can synergistically promote the growth of mulberry trees and the adsorption and transfer of heavy metal thallium, thereby improving the remediation efficiency of heavy metal thallium in soil. The effect is significantly better than that of single-strain Bacillus megaterium solution.

[0016] Furthermore, the compound microbial agent provided by this invention can be applied to the rhizosphere of mulberry trees by means of root irrigation with liquid microbial agent or by mixing with silkworm excrement organic fertilizer and then spreading it. This can further promote the growth of mulberry trees and improve the enrichment and transfer efficiency of the heavy metal thallium.

[0017] Furthermore, the method provided by this invention, through silkworm excrement organic fertilizer + compound microbial agent + mulberry tree, further promotes the growth of mulberry tree, accelerates the transfer and enrichment of heavy metal thallium, and fully leverages the effect of 1+1+1>3. It is a combined remediation method that can synergistically improve thallium remediation efficiency, regulate the distribution of thallium in mulberry tree, and ensure the safety of mulberry leaf feed, and has important practical significance and application value.

[0018] Biological Preservation Instructions Bacillus megaterium, taxonomic name is Bacillus megatherium It was deposited on May 10, 2017 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, with accession number GDMCC No: 60167.

[0019] Bacillus subtilis SEM-2, taxonomically named Bacillus ( ) Bacillus The specimen (sp.) was deposited on May 16, 2016 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, with accession number GDMCC No: 60039.

[0020] Bacillus cereus SEM-15, taxonomically named Bacillus cereus It was deposited on July 1, 2021 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, with accession number GDMCC No: 61562. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0022] Figure 1 The results show the soluble phosphorus content analysis in the SEM-4 fermentation broth after 96 hours of fermentation; different lowercase letters represent significant differences ( p <0.01); Figure 2 To jointly remediate the thallium content in rhizosphere soil at different times; different letters in the figure represent significant differences in samples at different times ( p <0.05); Figure 3The figure shows the thallium content in dried mulberry leaf powder; different letters in the figure represent significant differences in samples taken at different times. p <0.05); Figure 4 The remediation effects of different mulberry varieties on the heavy metal thallium in soil; different letters above each bar chart represent significant differences between different varieties at the same time. p <0.05). Detailed Implementation

[0023] This invention provides a compound microbial agent, the effective components of which include Bacillus subtilis SEM-2, Bacillus cereus SEM-15, and Bacillus megaterium; the preservation number of Bacillus subtilis SEM-2 is GDMCC No: 60039; the preservation number of Bacillus cereus SEM-15 is GDMCC No: 61562; and the preservation number of Bacillus megaterium is GDMCC No: 60167.

[0024] In one embodiment, the live count ratio of Bacillus subtilis SEM-2, Bacillus cereus SEM-15, and Bacillus megaterium is 1:1:1.

[0025] As one embodiment, the compound microbial agent has a live bacteria concentration ≥1×10⁻⁶. 9 Solid bacterial powder with CFU / g, or a live bacteria concentration ≥1×10⁻⁶ 9 Liquid bacterial agent at CFU / mL.

[0026] The Bacillus megaterium in the compound microbial agent provided by this invention is a strain isolated from the rhizosphere soil of mulberry trees, exhibiting significant phosphorus and potassium solubilization and heavy metal tolerance. Its characteristics are as follows: The colonies are milky white, round, with a smooth, slightly raised surface, opaque, and with neat edges. The strain exhibits good acid and alkali tolerance, growing within a pH range of 4-10, and is heat-resistant, surviving cultivation and transportation at 60℃. This gives it a significant advantage in the preparation of soil microbial inoculants. *Bacillus megaterium* can efficiently decompose inorganic phosphorus and potassium in the soil, activating soil nutrients and promoting plant growth. Especially since this strain was isolated from the rhizosphere soil of mulberry trees in areas contaminated with heavy metals, it demonstrates excellent synergy in heavy metal tolerance and mulberry growth promotion.

[0027] The compound microbial agent provided by this invention contains Bacillus megaterium, which originates from the rhizosphere soil of mulberry trees. It has a significant growth-promoting effect on mulberry trees and is highly adaptable to the environment with strong colonization ability. Bacillus subtilis SEM-2 and Bacillus cereus SEM-15 both come from silkworm excrement and are compatible with silkworm excrement organic fertilizer. Bacillus subtilis SEM-2 has excellent growth-promoting and disease-preventing functions, which can effectively promote the healthy growth of mulberry trees. Bacillus cereus SEM-15 has excellent heavy metal tolerance, adsorption and passivation ability, and plant growth-promoting ability. Both strains are well compatible with silkworm excrement organic fertilizer. When combined with Bacillus megaterium, they synergistically promote the growth of mulberry trees and the adsorption and transfer of heavy metal thallium, thereby improving the remediation efficiency of heavy metal thallium in the soil.

[0028] Based on the above advantages, the present invention provides the application of the composite microbial agent described in the above technical solution in one or more of the following: 1) Improve the survival rate of plants in thallium-contaminated soil; 2) Reduce the content of the heavy metal thallium in the soil; 3) Increase the activity of rhizosphere soil enzymes in thallium-contaminated soil; 4) Decompose inorganic phosphorus; 5) Promotes plant growth; 6) Reduce the content of the heavy metal thallium in plant leaves.

[0029] In one embodiment, the rhizosphere soil enzymes include one or more of acid phosphatase, urease, and catalase. In another embodiment, the plant includes a mulberry tree.

[0030] This invention provides a method for promoting plant growth and remediating thallium-contaminated soil, comprising the following steps: Mulberry trees were planted in thallium-contaminated soil, and the compound microbial agent described in the above technical solution was applied.

[0031] As one implementation method, the application concentration of the compound microbial agent is ≥3×10⁻⁶. 6 CFU / mL; the application method includes root drenching, which is performed once every 25-35 days, with 300-800 mL per plant each time. As one embodiment, the root drenching is performed once every 25-35 days, with 300-800 mL per plant each time. As another embodiment, the root drenching is performed once every 25-35 days, with 400-600 mL per plant each time. As yet another embodiment, the root drenching is performed once every 30 days, with 500 mL per plant each time.

[0032] In one embodiment, the method further includes: applying silkworm excrement organic fertilizer; the application rate of the silkworm excrement organic fertilizer is 0.5-2 tons / mu. In another embodiment, the application rate of the silkworm excrement organic fertilizer is 0.8-1.6 tons / mu. Silkworm excrement, as an abundant agricultural waste resource in the silkworm-producing areas of South China, is rich in organic matter and nitrogen, phosphorus, and potassium nutrients. After being applied to the soil, it can improve the physical and chemical properties of the soil and affect the transformation of heavy metals through adsorption and complexation. The compound microbial agent provided by this invention can be applied to the rhizosphere of mulberry trees by means of liquid microbial agent drenching or mixing with silkworm excrement organic fertilizer and then spreading it, which can further promote the growth of mulberry trees and improve the enrichment and transfer efficiency of the heavy metal thallium. In one embodiment, silkworm excrement organic fertilizer is applied as a top dressing every six months.

[0033] In one implementation method, the mulberry tree is Yue Sang 11. This invention has found that Yue Sang 11 exhibits significantly better absorption and enrichment effects on the heavy metal thallium than Yue Shen Da 10 and Yue Sang 51.

[0034] As one implementation method, a multi-point sampling method is used to determine the pH value of thallium-contaminated soil, and the initial pH is adjusted to 6.5-7.5. As another implementation method, the method of the present invention is applicable to acidic heavy metal contaminated soils in pyrite mining areas.

[0035] To further illustrate the present invention, the following detailed description, in conjunction with embodiments and accompanying drawings, describes the application of the compound microbial agent provided by the present invention and its combination with mulberry and silkworm excrement organic fertilizer in the remediation of thallium-contaminated soil. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1: Identification of the ability of Bacillus megaterium to solubilize inorganic phosphorus Activate Bacillus megaterium (SEM-4 strain), inoculate into NB medium at a 1% (v / v) inoculation rate, and incubate overnight at 37°C with shaking. The bacterial concentration is approximately 2-3 × 10⁻⁶. 8 CFU / mL was inoculated into inorganic phosphorus liquid medium at a 1% (v / v) inoculum and co-fermented, with commercially available phosphate-solubilizing bacteria powder as a control. After culturing at 37℃ and 150 rpm for 96 h with shaking, the available phosphorus content in the fermentation supernatant was determined using the molybdenum antimony colorimetric method. The results showed that the soluble phosphorus content in the fermentation broth of strain SEM-4 of this invention reached 184 mg / L, significantly higher than the 90.75 mg / L result of the commercially available phosphate-solubilizing bacteria powder. Figure 1 ).

[0037] Example 2: Identification of the adsorption of heavy metal thallium by Bacillus megaterium After activating the SEM-4 strain and the concurrently screened Bacillus strains S2, S5, and S8, they were inoculated into NB medium at a 1% (v / v) inoculation rate and incubated overnight at 37°C with shaking. The strain concentration was approximately 2-3 × 10⁻⁶. 8CFU / mL, different bacterial suspensions were prepared with thallium standard solution to achieve a bacterial concentration of 10. 8 A mixed culture medium with CFU / mL and a final thallium concentration of 10 mg / L was prepared, mixed well, and allowed to stand for 5-6 hours. The mixture was then centrifuged at 4000 rpm for 5 minutes, and the supernatant was collected. The content of soluble thallium in the supernatant was then determined. The results are shown in Table 1.

[0038] Table 1. Analysis of adsorption efficiency of different strains for heavy metal thallium.

[0039] Note: Different lowercase letters indicate significant differences. p <0.01); Adsorption efficiency % = (Thallium concentration in the supernatant of the blank group - Thallium concentration in the supernatant after adsorption of each group) / Thallium concentration in the supernatant of the blank group × 100%.

[0040] The results showed that the adsorption efficiency of the SEM-4 strain of the present invention for the heavy metal thallium reached 30.6%, which was significantly higher than that of the other three Bacillus strains.

[0041] Example 3: Preparation of SEM-4 bacterial raw powder and compound bacterial powder SEM-4 strain was activated, a single colony was picked and inoculated into 100 ml of NB liquid medium (sterilized at 121℃ for 20 min), and cultured at 37℃ and 200 r / min for 14 h to obtain primary seed culture.

[0042] The primary seed culture was inoculated into a sterile seed tank at an inoculation rate of 0.7–1% (v / v). The culture conditions were: aeration ratio of 1:1, stirring speed of 150–250 rpm, culture temperature of 37°C, tank pressure of 0.05 MPa, and culture time of 10 h. At the end of the culture, a secondary seed culture was obtained, with a viable bacterial content of approximately 10%. 7 CFU / mL, OD value of 0.8~0.9, at which point the bacterial strain has strong viability.

[0043] SEM-4 bacterial fermentation The secondary seed culture of SEM-4 was inoculated into a sterile fermenter at an inoculation rate of 0.5-1% (v / v). The culture conditions were: aeration ratio of 1:(1-1.5), stirring speed of 200-300 r / min, culture temperature of 32-37℃, fermenter pressure of 0.05 MPa, maintaining dissolved oxygen levels above 5%, and culture time of approximately 50 hours. The fermentation was completed when over 90% of the spores were released, as determined by microscopic examination of samples. The viable SEM-4 spore content in the mature fermentation broth was 3-4 × 10⁻⁴ spores. 9 CFU / mL was used to obtain a mature fermentation broth.

[0044] Preparation of SEM-4 raw powder The fermentation mature bacterial broth obtained above (3~4×10)9 The original powder of SEM-4 strain (CFU / mL) was obtained by concentration and spray drying.

[0045] The preparation methods of bacterial suspension and powder of Bacillus subtilis (GDMCC No: 60039) SEM-2 and Bacillus cereus SEM-15 (GDMCC No: 61562) are based on the relevant methods in patents CN201910730214.7 and CN202210052408.8.

[0046] Preparation of compound microbial agents The SEM-4 raw powder obtained above was mixed with the raw powders of Bacillus subtilis SEM-2 and Bacillus cereus SEM-15 at a ratio of 1:1:1 to obtain the composite microbial agent raw powder.

[0047] Example 4: Effects of different microbial inoculants on mulberry tree growth and soil remediation in thallium-contaminated areas. SEM-4 raw powder and compound microbial inoculant raw powder were prepared using the method in Example 3. The different raw powders were dissolved in water to obtain a viable bacteria concentration of 10. 6 SEM-4 bacterial suspensions and compound bacterial suspensions with CFU / mL were subjected to the following grouping treatments: Group 1 (SEM-4 bacterial solution): One week after planting mulberry seedlings in pots of soil from the mining area (thallium content 7.6 mg / kg soil, the same below), 10 mg / kg of the solution was applied to the root zone of each pot for root irrigation. 6 Apply 500 mL of SEM-4 bacterial solution with a concentration of CFU / mL to the roots, followed by root irrigation every 30 days. Group 2 (SEM-4 + silkworm excrement organic fertilizer): Mulberry seedlings were planted after mixing the mining area soil with silkworm excrement organic fertilizer (purchased from Guangdong Plant Dragon Biotechnology Co., Ltd.) at a mass ratio of 19:1. 10g of the mixture was applied to the root zone of each seedling. 6 Apply 500 mL of SEM-4 bacterial solution with a concentration of CFU / mL to the roots, followed by root irrigation every 30 days. Group 3 (compound microbial agent): One week after planting mulberry seedlings in pots in the mining area soil, apply 10g of the agent to the root zone of each pot. 6 Apply 500 mL of the compound bacterial solution with a concentration of CFU / mL to the roots, and then drench the roots every 30 days thereafter. Group 4 (compound microbial agent + silkworm excrement organic fertilizer): Mulberry seedlings were planted after mixing the mining area soil with silkworm excrement organic fertilizer (purchased from Guangdong Plant Dragon Biotechnology Co., Ltd.) at a mass ratio of 19:1. 10g of the mixture was applied to the root zone of each pot for root irrigation. 6 Apply 500 mL of the compound bacterial solution with a concentration of CFU / mL to the roots, and then drench the roots every 30 days thereafter. Group 5 (blank control): Mulberry seedlings were planted in soil basins in the mining area, without the application of fungicides or silkworm excrement organic fertilizer.

[0048] After 270 days, the survival rate of mulberry seedlings in pots, changes in soil thallium content (graphite furnace atomic absorption spectrophotometry), and changes in soil rhizosphere enzyme activity (Nanjing Jiancheng reagent kit) were measured in different treatment groups. The results are shown in Table 2.

[0049] Table 2. Effects of different treatment groups on the cultivation of mulberry seedlings in potted soil contaminated with thallium.

[0050] Note: Different lowercase letters indicate significant differences. p <0.05).

[0051] The results showed that, in terms of mulberry seedling survival rate, thallium transfer efficiency in the soil 180 days after planting, and changes in the activity of acid phosphatase, urease, and catalase in the rhizosphere soil, all four treatment groups showed significant improvement compared to the blank control group, with the fourth group (compound microbial agent + silkworm excrement organic fertilizer + mulberry tree) showing the best effect.

[0052] Example 5 A method for the co-remediation of heavy metals based on silkworm excrement, microorganisms, and mulberry trees includes: Soil pretreatment: Multiple samples were taken from the surface soil of the mining area to investigate the pollution level of the heavy metal thallium and the soil pH. If the soil was too acidic (pH < 5.0), an appropriate amount of lime was applied to adjust the initial pH to 6.5-7.5.

[0053] Silkworm excrement collection and fermentation: Collect the organic waste silkworm excrement from surrounding mulberry planting and silkworm rearing, and carry out static aerobic composting fermentation treatment. The fermentation time is ≥30 days. Turn the pile once every 10-15 days of fermentation. Fermentation ends after the temperature in the middle of the fermentation pile returns to normal.

[0054] Compound preparation of microbial agents: Compound microbial agent raw powder was prepared by the method in Example 3.

[0055] Joint remediation: Fermented and decomposed silkworm excrement organic fertilizer is mixed with silkworm excrement biochar at a mass ratio of 4:1 and then evenly applied to the contaminated soil at a rate of 2 tons / acre. Deep plowing and compaction are performed, creating wide ridges: ridge width 60cm, ridge spacing 80cm. Select mulberry seedlings of Yue Sang 51 (a variety with strong heavy metal tolerance and significant accumulation effect) and plant them on both sides of the wide ridges: row spacing 60cm, plant spacing 30cm. Water thoroughly to settle the roots. One week later, dilute the compound microbial agent powder with water to a concentration of 3×10⁻⁶. 6 A compound bacterial solution of CFU / mL was applied to the rhizosphere soil of each mulberry seedling using the root-potting method, with 500mL of the compound bacterial solution applied to the rhizosphere soil.

[0056] Field management: Water regularly according to the weather (water once every 15-20 days if it does not rain; if it rains or the humidity is high, watering is not necessary). Apply 500mL of compound bacterial solution every month to promote the growth of mulberry trees and the reproduction of microorganisms. Apply fermented and decomposed silkworm excrement organic fertilizer every six months. Weed regularly, control pests, and prune mulberry seedlings regularly.

[0057] Example 6 Using the spoil heap of the Guangdong Yunliu Mining Area as the experimental area, with an initial soil pH of 7.15, the method of Example 5 was employed for remediation. Based on the initial thallium content of the soil in the experimental area, samples were collected at 90, 180, and 270 days after mulberry seedling transplantation. Rhizosphere soil samples were collected from the top 5-10 cm of soil, with three replicates for each experimental area. The soil was dried in a cool, shaded place for later use. Mulberry leaves were sampled at 90, 180, and 270 days after seedling establishment, dried, and pulverized for later use. Soil and mulberry leaf powder samples were analyzed using atomic absorption spectrophotometry as specified in the relevant standard HJ / 1080-2019.

[0058] Soil test results as follows Figure 2 As shown in the figure. The results show that the background value of thallium content in the topsoil of the pyrite mining area is high, greater than 7 mg / kg. Its deposition and transfer with surface water will pose a very high safety risk to the surrounding soil and water sources. After the combined remediation using silkworm excrement-microorganisms + mulberry trees, the thallium content in the soil showed a significant decreasing trend. p <0.05), the background value dropped to about 4.1 mg / kg after 270 days, with a remediation efficiency of 44.59%. With the extension of time, it is predicted that the thallium content in the soil of the test area will drop to below 2.0 mg / kg in 2-3 years.

[0059] The results of the determination of thallium content in mulberry leaf powder are shown below. Figure 3 The results showed that during the period of 90-270 days after planting, the thallium content in the dry matter of mature mulberry leaves was measured, and it was found that the thallium content in the mulberry leaves decreased significantly with the extension of time. p <0.05), and after 180 days, the thallium content in mulberry leaf powder decreased to below 0.3 mg / kg.

[0060] Comparative Example 1 The method is similar to that in Example 6, except that the fermented and decomposed silkworm excrement is replaced with pond mud or sludge. The pond mud comes from the bottom of surrounding fish ponds. The mud is dried and crushed, with a pH of 7.05 and an organic matter content of 13.8%. The sludge is obtained from a mining company, with a pH of 7.3 and an organic matter content of >30%.

[0061] Each group underwent three parallel replicate experiments, with sampling and management methods identical to those in Example 6. Rhizosphere soil samples were collected at different remediation times, and the thallium content was determined.

[0062] The results showed that after 270 days of remediation treatment, the thallium content in the rhizosphere soil of the silkworm excrement group decreased from 7.40 mg / kg to 4.10 mg / kg, a reduction of 44.6%; the pond mud group decreased from the initial 7.10 mg / kg to 6.33 mg / kg, with a remediation rate of 10.8%; and the sludge group decreased from the initial 7.35 mg / kg to 6.63 mg / kg, with a remediation rate of 9.8%. The remediation efficiency of both the pond mud and sludge groups was significantly lower than that of the silkworm excrement group. p <0.01).

[0063] Comparative Example 2 To select mulberry varieties with better combined remediation effects with silkworm excrement and microorganisms, three mulberry resource varieties with different uses, namely Yuezhenda 10, Yuesang 11 and Yuesang 51, were selected for comparative analysis.

[0064] Referring to the four methods in Example 4, soil from the mining area was used as potting substrate. Silkworm excrement organic fertilizer was mixed in at a mass ratio of 19:1, and the mixture was then divided into pots. Mulberry seedlings were planted in 15 pots (plants) of each variety. Soil samples were collected at 90, 180, and 270 days after planting, and newly sprouted mulberry branches were pruned at the same time. The content of the heavy metal thallium in the potting soil was tested according to the method in Example 6.

[0065] The results are as follows Figure 4 As shown, all three mulberry varieties have the effect of absorbing and accumulating the heavy metal thallium, among which Yue Sang 11 has a significantly better effect than the other two varieties.

[0066] Comparative Example 3 To further clarify the effects of different combinations on the transfer and enrichment of the heavy metal thallium in soil, three groups were established: silkworm excrement group (1), silkworm excrement + mulberry tree group (2), and silkworm excrement + microorganisms + mulberry tree group (3), as detailed below: No. 1: Take soil from the mining area as potting substrate, mix it with silkworm excrement organic fertilizer at a mass ratio of 19:1, and then divide it into pots, unplanted mulberry seedlings, and root irrigation compound bacterial solution. No. 2: Similar to No. 1, except that it is planted with Yue Sang No. 11 mulberry seedlings; No. 3: Similar to No. 1, the difference is that it involves planting Yue Sang No. 11 mulberry seedlings and applying compound bacterial solution to the roots; The potted plants were managed in the same way as control group 2. After 270 days, rhizosphere soil samples were taken to analyze the total content of the heavy metal thallium in the soil. The results are shown in Table 3.

[0067] Table 3. Transfer and enrichment effects of different groups on thallium in soil.

[0068] The results showed that the total amount of thallium in the soil did not change significantly in the silkworm excrement group alone. This may be because after in-situ adsorption by the silkworm excrement, the thallium was transferred from the soil to the pores of the excrement, changing its absorbability or bioavailability by plants, but without significantly changing its total amount. In the silkworm excrement + mulberry tree group, due to plant absorption and transfer, the total thallium content in the soil decreased from 6.4 mg / kg to 4.6 mg / kg, with a remediation efficiency of 28%. This may be because the thallium in the soil was transferred after being absorbed and enriched by the roots of mulberry trees, significantly reducing the thallium content in the soil. The silkworm excrement + microorganisms + mulberry tree group had the highest remediation efficiency, exceeding 40%. This may be because the combination of silkworm excrement organic fertilizer and compound microbial inoculant promoted the growth of mulberry trees, accelerated the transfer and enrichment of heavy metal thallium, and fully exerted the effect of 1+1+1>3.

[0069] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A compound microbial agent, characterized in that, The active ingredients include Bacillus subtilis ( Bacillus sp.) SEM-2, Bacillus cereus ( Bacillus cereus SEM-15 and Bacillus megaterium ( Bacillus megatherium The Bacillus subtilis SEM-2 has a GDMCC No. 60039; the Bacillus cereus SEM-15 has a GDMCC No. 61562; and the Bacillus megaterium has a GDMCC No. 60167.

2. The compound microbial agent according to claim 1, characterized in that, The viable cell ratio of Bacillus subtilis SEM-2, Bacillus cereus SEM-15, and Bacillus megaterium was 1:1:

1.

3. The compound microbial agent according to claim 1 or 2, characterized in that, The compound microbial agent has a viable bacteria concentration ≥1×10⁻⁶. 9 Solid bacterial powder with CFU / g, or a live bacteria concentration ≥1×10⁻⁶ 9 Liquid bacterial agent at CFU / mL.

4. The use of the compound microbial agent according to any one of claims 1-3 in one or more of the following: 1) Improve the survival rate of plants in thallium-contaminated soil; 2) Reduce the content of the heavy metal thallium in the soil; 3) Increase the activity of rhizosphere soil enzymes in thallium-contaminated soil; 4) Decompose inorganic phosphorus; 5) Promotes plant growth; 6) Reduce the content of the heavy metal thallium in plant leaves.

5. The application according to claim 4, characterized in that, The rhizosphere soil enzymes include one or more of acid phosphatase, urease, and catalase.

6. The application according to claim 4 or 5, characterized in that, The plants mentioned include mulberry trees.

7. A method for promoting plant growth and remediating thallium-contaminated soil, characterized in that, Includes the following steps: Mulberry trees are planted in thallium-contaminated soil, and the compound microbial agent described in any one of claims 1-3 is applied.

8. The method according to claim 7, characterized in that, The application concentration of the compound microbial agent is ≥10. 6 CFU / mL; the application method includes root irrigation, which is performed once every 25-35 days, with 300-800 mL per plant each time.

9. The method according to claim 7, characterized in that, The method further includes: applying silkworm excrement organic fertilizer; the application rate of the silkworm excrement organic fertilizer is 0.5-2 tons / mu.

10. The method according to any one of claims 7-9, characterized in that, The mulberry tree in question is Yue Sang No. 11.

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