A copper mine tailing pond heavy metal contaminated soil ecological treatment method based on a composite modifier-bacillus subtilis-baishoushan carex

Abstract

This invention specifically relates to an ecological remediation method for heavy metal-contaminated soil in copper mine tailings ponds based on a composite amendment—Bacillus subtilis—and *Carex balsamina*, belonging to the field of ecological remediation technology for heavy metal pollution in mining soils. The method includes soil pretreatment, preparation and mixing of the composite amendment, addition of Bacillus subtilis bacterial solution, and planting and maintenance of *Carex balsamina*. The composite amendment, with a specific ratio, rapidly improves the physicochemical properties of the highly acidic and barren tailings soil, creating a suitable habitat for functional microorganisms and resilient plants. Bacillus subtilis activates rhizosphere heavy metals and promotes plant growth, forming a symbiotic and synergistic system with *Carex balsamina*, thereby enriching the soil with heavy metals such as Cu and Cd. This invention significantly increases soil pH, reduces salinity, increases organic matter, and improves the removal rate of Cu and Cd. The method uses readily available raw materials, is simple to operate, and is environmentally friendly, making it suitable for large-scale ecological remediation of heavy metal-contaminated sites such as copper mine tailings ponds.

Description

Technical Field

[0001] This invention belongs to the field of ecological remediation technology for heavy metal pollution in mine soil, specifically involving an ecological remediation method for heavy metal polluted soil in copper mine tailings ponds based on a composite modifier-Bacillus subtilis-Carex balsamina. Background Technology

[0002] Tailings ponds generated during mining operations commonly suffer from high acidity, low fertility, and excessive levels of heavy metals (Cu, Zn, Pb, Cd, etc.). Heavy metals in soil exhibit irreversible and bioaccumulative properties, damaging soil ecological structure, inhibiting plant growth, and ultimately threatening ecosystem stability and human health through the food chain. Current mine heavy metal pollution remediation technologies mainly include physical remediation (covering and isolation, dry tailings discharge), chemical remediation (solidification / stabilization, leaching), and bioremediation (phytoremediation, microbial activation / fixation). Physicochemical methods are costly and prone to secondary pollution, while single bioremediation technologies are inefficient and time-consuming. However, combined microbial-phytoremediation-soil conditioner technologies have become a research hotspot due to their environmental friendliness, low cost, and strong sustainability. Nevertheless, there are still few dedicated composite remediation systems for typical mine-contaminated soils such as copper mine tailings ponds, and the lack of clearly defined optimal parameters and standardized operating procedures limits their engineering application.

[0003] Microbial-phytoremediation-soil amendment co-remediation technology has become a research hotspot due to its environmental friendliness, low cost, and strong sustainability. However, for special soils with multi-metal complex pollution, primarily copper, existing technologies still lack efficient, stable, and parameter-defined synergistic remediation systems. For example, conventional lime amendments may exacerbate soil compaction; single-plant remediation has low survival rates and long remediation cycles in extremely acidic and infertile tailings soils; and the activity of common rhizosphere growth-promoting bacteria is easily inhibited under strong acid and high-metal stress.

[0004] Therefore, there is an urgent technical need and engineering application value in developing a standardized remediation method for copper mine tailings ponds that, through the selection and optimization of specific components, can rapidly improve the soil's physical and chemical properties, significantly promote plant-microbe symbiosis, and efficiently and synergistically remove heavy metals. Summary of the Invention

[0005] The purpose of this invention is to address the problems of insufficient targeting and unclear synergistic efficiency in the existing technology for soil remediation of copper mine tailings ponds. It provides an ecological remediation method based on a specific composite amendment, Bacillus subtilis, and sedge in synergy. By clarifying the optimal ratio of each component and the operation process, it can achieve rapid improvement of the physical and chemical properties of this type of soil and efficient and synergistic removal of heavy metals.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] An ecological remediation method for heavy metal contaminated soil in copper mine tailings ponds based on a composite amendment-Bacillus subtilis-Carex balsamina, comprising the following steps:

[0008] 1) Pretreatment of contaminated soil: Collect heavy metal contaminated soil from copper mine tailings pond at a depth of 10-30cm, remove stones and waste branches and leaves, air dry naturally, and then crush and pass through a 20-mesh sieve for later use.

[0009] 2) Preparation of composite amendment: Dry coconut coir and peat moss separately, cool them and mix them evenly to prepare a composite amendment;

[0010] 3) Preparation of improved soil matrix: Mix the composite amendment with the pretreated contaminated soil. The mass ratio of the composite amendment is 25% to 45%. Stir evenly to form improved soil matrix.

[0011] 4) Addition of functional microorganisms: Add Bacillus subtilis bacterial solution to the improved soil substrate and mix well;

[0012] 5) Planting: Select healthy seedlings of *Carex balsamina* and plant them in the improved soil obtained in step 4);

[0013] 6) Maintenance and management: After planting, replenish water regularly to maintain suitable soil moisture. The cultivation period is 100 days. In the middle of the planting period, supplement with Bacillus subtilis inoculum solution and in the later stage of planting, supplement with nitrogen fertilizer.

[0014] Furthermore, in the above-mentioned method for ecological remediation of heavy metal contaminated soil in copper mine tailings ponds based on a composite modifier-Bacillus subtilis-Carex balsamina, step 2) involves drying at 105°C for 8 hours.

[0015] Furthermore, in the above-mentioned method for ecological remediation of heavy metal contaminated soil in copper mine tailings ponds based on a composite amendment-Bacillus subtilis-Carex balsamina, in step 2), coconut coir and peat soil are mixed at a mass ratio of 1:1.

[0016] Furthermore, in the above-mentioned method for ecological remediation of heavy metal contaminated soil in copper mine tailings ponds based on composite amendment-Bacillus subtilis-Carex balsamina, in step 3), the mass ratio of the composite amendment is 35%.

[0017] Furthermore, in the above-mentioned ecological remediation method for heavy metal contaminated soil in copper mine tailings ponds based on a composite amendment-Bacillus subtilis-Carex balsamina, in step 4), the effective viable count of the Bacillus subtilis bacterial solution is 1×10⁻⁶. 8 CFU~1×10 9The concentration of CFU / mL and the addition amount of Bacillus subtilis bacterial solution is 0.2 mL per 0.3 kg of improved soil substrate.

[0018] Furthermore, in the above-mentioned method for ecological remediation of heavy metal contaminated soil in copper mine tailings ponds based on a composite amendment-Bacillus subtilis-Carex balsamina, in step 6), the mid-term after planting is the 15th to 30th day after planting, and the late-term after planting is the 30th to 50th day after planting.

[0019] Furthermore, in the above-mentioned ecological remediation method for heavy metal contaminated soil in copper mine tailings ponds based on a composite amendment-Bacillus subtilis-Carex balsamina, in step 6), the effective viable count of the Bacillus subtilis bacterial solution is 1×10⁻⁶. 8 CFU~1×10 9 The concentration of CFU / mL and the addition amount of Bacillus subtilis bacterial solution is 0.2 mL per 0.3 kg of improved soil substrate.

[0020] Furthermore, in the above-mentioned method for ecological remediation of heavy metal contaminated soil in copper mine tailings ponds based on a composite amendment-Bacillus subtilis-Carex balsamina, in step 6), the nitrogen fertilizer is urea.

[0021] The beneficial effects of this invention are as follows:

[0022] 1. Targeted Improvement and Synergistic Effect: This invention employs a specific composite soil conditioner, a 1:1 mixture of coconut coir and peat moss, to address the problems of strong acidity, infertility, and compaction in tailings pond soils, producing a synergistic improvement effect. Coconut coir improves soil permeability, while peat moss provides organic matter and fine-tunes pH. Experiments show that after incorporating 35% of this conditioner, the soil pH can be increased from 4.725 to 4.902, and further stabilized at 5.712 after plant-microbe combined action; electrical conductivity decreases by approximately 11.6%, and further decreases by 42.3% after plant-microbe combined action, for a cumulative reduction of 48.7%; organic matter content increases by over 159.6%, creating an unpredictable and suitable microenvironment for subsequent bioremediation.

[0023] 2. Prominent Microbial-Plant Symbiotic Effect: In the soil environment optimized by the compound amendment, Bacillus subtilis and *Carex balsamina* formed a significant symbiotic synergistic effect. As a rhizosphere growth-promoting bacterium (PGPR), Bacillus subtilis not only secretes growth-promoting substances but also exhibits enhanced activity in the microenvironment created by the amendment, interacting with the well-developed root system of *Carex balsamina* to jointly activate and absorb heavy metals. Experimental data show that the specific combination of this invention, with a 35% amendment ratio, increased the fresh weight of the underground parts of *Carex balsamina* by 159.4% compared to the 15% amendment group. Its removal rates of Cu and Cd in the soil reached 53.73% and 87.03%, respectively, and the enrichment of Cu in the underground parts of the plant reached as high as 1911.94 mg / kg, far exceeding the expectations of individual components or simple superposition.

[0024] 3. Optimal parameters are clearly defined and not obvious: Through systematic pot experiments, this invention determined the optimal incorporation ratio of the composite soil conditioner to be 35%. This ratio represents the best balance between soil pH adjustment, salinity reduction, organic matter enhancement, plant biomass accumulation, and total heavy metal extraction, and cannot be obtained through simple linear gradient experiments. When the ratio is lower or higher than this value, the aforementioned synergistic effects significantly decrease, demonstrating the criticality and non-obviousness of this specific parameter in achieving the technical effects of this invention.

[0025] 4. The remediation system has strong adaptability and significant synergistic effect: This invention addresses the core problems of "acidity, poorness, and toxicity" in copper mine tailings pond soil. It proposes and verifies for the first time a specific combination of "coconut coir-peat soil 1:1 composite conditioner + Bacillus subtilis + sedge". Through functional complementarity and temporal coupling, it achieves a synergistic remediation effect of "1+1+1>3", which is far superior to single technologies or simple combinations.

[0026] 5. Comprehensive and lasting improvement in soil physicochemical properties: The 35% compound amendment can stably raise the pH of strongly acidic tailings soil to the optimal growth range of Paeonia suffruticosa (5.0~6.5), reduce the electrical conductivity to the suitable salinity range for plants, and significantly increase organic matter, effectively improving soil aggregate structure and fertilizer and water retention capacity, laying a stable foundation for bioremediation.

[0027] 6. Excellent heavy metal removal and enrichment efficiency: Under optimal parameters, the removal rates of Cu and Cd reach 53.73% and 87.03%, respectively. The Cu enrichment in the underground part of the Baitoushan moss is as high as 1911.94 mg / kg. It also has stable removal and enrichment effects on Zn and Pb. Its comprehensive remediation performance is better than most existing combined remediation technologies.

[0028] 7. High value for engineering application and promotion: The raw materials used are all commercially available products with low cost; the operation process is simple and clear, easy to standardize and implement on a large scale; the remediation process has no secondary pollution, is environmentally friendly, and is suitable for large-scale ecological remediation of heavy metal contaminated sites such as copper mine tailings ponds. Attached Figure Description

[0029] Figure 1 A roadmap for ecological remediation of heavy metal contaminated soil in copper mine tailings ponds.

[0030] Figure 2 The effect of different proportions of compound amendment on the plant height of *Carex balsamina*.

[0031] Figure 3 The effect of different proportions of compound modifier on the number of leaves of *Carex balsamina*.

[0032] Figure 4 The effect of different proportions of compound modifier on the dry and fresh weight of the aboveground parts of the Baitoushan sedge.

[0033] Figure 5 The effect of different proportions of composite modifiers on the dry and fresh weight of the understory of moss from Mount Baitou. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the embodiments of this invention will be described in detail below with reference to examples. The following examples are for illustrative purposes only and should not be considered as limiting the scope of this invention.

[0035] Example 1

[0036] (I) An ecological remediation method for heavy metal contaminated soil in copper mine tailings ponds based on a composite amendment-Bacillus subtilis-Carex balsamina

[0037] 1) Soil Sampling and Pretreatment: Soil was collected from the surface of the tailings pond of the Fushun Copper Mine in areas without vegetation growth (sampling depth 10-30cm). Stones, weed roots, and other impurities were removed, and the soil was air-dried and then pulverized through a 20-mesh sieve. Initial physicochemical properties were determined: pH 4.725, conductivity 3.36 mS / cm, organic matter content 0.994 g / kg, Cu content 158.84 mg / kg, Zn content 250.83 mg / kg, Pb content 1659.21 mg / kg, and Cd content 289.07 mg / kg.

[0038] 2) Preparation of composite amendment: Coconut coir and peat moss were dried at 105℃ for 8 hours, cooled and then mixed evenly at a mass ratio of 1:1 to prepare composite amendment.

[0039] 3) Preparation of soil substrate: The composite soil conditioner was added at the following mass ratios: 0% (control group), 15%, 25%, 35%, and 45%, respectively. The mixture was then mixed evenly with the pretreated contaminated soil and placed into plastic flower pots with bases at the bottom. Each pot contained 0.3 kg of soil, and each treatment was set up in 3 replicates.

[0040] 4) Addition of Bacillus subtilis: Add Bacillus subtilis (B. subtilis) to the flowerpots of each treatment group. Bacillus subtilis Bacterial solution (effective viable count of 5 × 10⁻⁶) 8 The dosage is 0.2 mL of bacterial solution (total viable count 1×10⁻⁶ CFU / mL) per 0.3 kg of improved soil substrate. 8 CFU), mix well.

[0041] 5) Planting of *Carex balsamina*: Select *Carex balsamina* plants with uniform growth (… Carexpeiktusani Seedlings, prune the above-ground parts to a height of 5cm, plant 4 seedlings per pot, place them indoors for cultivation, and spray water on the leaves daily during the seedling establishment period.

[0042] 6) Maintenance and management: Replenish Bacillus subtilis bacterial solution once on the 17th and 27th day after planting (addition amount is the same as in step 4); apply 1.14g of urea on the 33rd and 46th day after planting; replenish deionized water regularly to maintain suitable soil moisture, and the cultivation cycle is 100 days.

[0043] (II) Indicator Measurement and Results

[0044] Table 1. Effects of soil conditioner mass ratio before and after planting on soil pH, electrical conductivity, and organic matter.

[0045]

[0046] Note: Different lowercase letters in the same column indicate differences at the 5% significance level (P<0.05); all data are the mean ± standard error of three parallel replicates.

[0047] Soil physicochemical properties: Table 1 shows that the application of the compound soil conditioner had a significant and dose-dependent effect on soil properties. Regarding pH, the original soil in the control group was strongly acidic (pH=4.725), severely unsuitable for plant growth. With increasing proportions of the conditioner, the soil pH before planting increased significantly (P<0.05), reaching 4.902 in the 35% group. After plant-microbe interaction, the pH further increased to 5.712 after planting, which falls within the optimal growth range for *Carex balsamina* (5.0~6.5), effectively and persistently alleviating soil acidification. The pH in the 45% group rose to 6.003 after planting, still within the suitable range, but did not result in a better plant growth response. Electrical conductivity analysis showed that the original soil salinity in the control group was too high (3.36 mS / cm), far exceeding the suitable range for plants (0.5~2.5 mS / cm). Soil amendment treatment effectively reduced soil electrical conductivity. In the 35% group, the electrical conductivity before planting decreased to 2.97 mS / cm, a reduction of 11.6% compared to the original soil. After plant-microorganism combined action, it further decreased to 1.713 mS / cm after planting, a reduction of 42.3% compared to the amended soil before planting, and a cumulative reduction of 48.7% compared to the original soil, fully entering the salinity range suitable for plant growth. Notably, the electrical conductivity of all amendment-treated groups was significantly lower than that of the control group (P<0.05), and the decrease increased with the increase of the amendment proportion. Regarding organic matter content, the soil amendment significantly improved soil fertility. The 35% amendment group had an organic matter content of 2.580 g / kg before planting, a substantial increase of 159.6% compared to the original soil (0.994 g / kg) (P<0.05). After one growth cycle, although the organic matter content decreased somewhat due to plant absorption and utilization, it still remained at a relatively high level of 2.233 g / kg. The increase in organic matter not only improved soil aggregate structure and enhanced water and fertilizer retention capacity, but also helped reduce the bioavailability of heavy metals through adsorption and complexation, mitigating their toxicity to plants and microorganisms. Overall, the 35% amendment ratio achieved the best balance between regulating soil pH, alleviating salt stress, and improving soil fertility, creating the most suitable microenvironment for subsequent plant establishment and microbial colonization.

[0048] Plant growth indicators: The measured height of the sedge is its natural height. In the early stages of planting, the sedge leaves are upright, and changes in plant height can well illustrate the growth status of the sedge. Figure 2 However, as time went on, the leaves of the sedge began to droop after it reached a certain height, and the natural height could no longer reflect the plant's growth status. Therefore, the measurement of plant height was stopped, and the measurement of the number of leaves was added instead. Figure 3 ).Depend on Figures 2 to 5It can be seen that, in terms of plant height and leaf number, the control group of *Carex balsamina* died completely in the middle of the experiment due to excessive soil acidity and salinity; while the plants in each amendment treatment group grew normally, with the 35% group showing the best growth, reaching a plant height of 16.4 cm, which was 27.4%, 31.7%, and 20.1% higher than the 15%, 25%, and 45% groups, respectively. Figure 2 The number of leaves reached 29, significantly higher than other treatment groups (P<0.05). Figure 3 This indicates that the soil environment at this ratio is most conducive to plant photosynthesis and nutrient absorption. Regarding biomass accumulation, the aboveground fresh weight (4.44g) and dry weight (2.16g) of the 35% ratio group were significantly higher than those of the previous year. Figure 4 The fresh weight (7.36g) and dry weight (4.67g) of the underground part (the underground part) Figure 5 Both were the highest among all treatment groups, with aboveground fresh weight increasing by 82.0% and underground dry weight increasing by 63.9% compared to the 25% group. Of particular note is that its underground dry weight reached 2.16 times that of the aboveground dry weight, indicating that *Carex balsamina* has a well-developed root system, which provides sufficient interfacial conditions for heavy metal absorption.

[0049] Table 2. Heavy metal content in soil before and after remediation under different proportions of composite amendment.

[0050]

[0051] Note: In the table, "before remediation" refers to the baseline value of heavy metals in the soil after the compound amendment is fully mixed with the corresponding proportion of contaminated soil, before inoculation with Bacillus subtilis and planting of Paeonia suffruticosa; "after remediation" refers to the final value of heavy metals in the soil after inoculation with Bacillus subtilis and planting of Paeonia suffruticosa in the corresponding proportion of soil matrix before remediation, and after 100 days of cultivation.

[0052] Heavy metal removal and enrichment effects: Table 2 shows that for Cu, the removal rate of the 35% group reached 53.73%, significantly higher than that of the 15% group (25.39%) and the 25% group (39.39%), and the 35% group showed better phytoaccumulation effect. For Cd, the 35% group had the highest removal rate (87.03%), significantly better than other treatment groups (P<0.05), indicating that this ratio can effectively promote the absorption of Cd by plants. Regarding Pb removal, the efficiency of each treatment group was generally low, with the 35% group only achieving 4.29%. This is mainly due to the poor mobility of Pb in the soil and its difficulty in being absorbed by plants, but the soil conditioner still improved the soil environment to some extent, thus enhancing the enrichment of Pb by plants. For Zn, the 35% group had the highest removal rate (45.48%), significantly higher than other treatment groups. Meanwhile, in terms of heavy metal enrichment, the underground accumulation of Cu, Pb, and Zn all reached their maximum values ​​at a 35% incorporation ratio. Although the underground concentration of Cd was highest at a 25% incorporation ratio (36.44 mg / kg), the plant biomass in the 35% group was significantly higher, resulting in a higher total Cd extraction (130.5 mg) than that in the 25% group (107.1 mg). Therefore, the 35% group had the highest soil removal rate for Cd.

[0053] Table 3. Content of four heavy metals in the underground parts of plants treated with the compound amendment

[0054]

[0055] Note: During the experiment, the sedges growing on the soil without the addition of the compound conditioner died and could not be measured, so they were not included.

Claims

1. A method for ecological remediation of heavy metal contaminated soil in copper mine tailings ponds based on a composite amendment-Bacillus subtilis-Carex balsamina, characterized in that, Includes the following steps: 1) Pretreatment of contaminated soil: Collect heavy metal contaminated soil from copper mine tailings pond at a depth of 10-30cm, remove stones and waste branches and leaves, air dry naturally, and then crush and pass through a 20-mesh sieve for later use. 2) Preparation of composite amendment: Dry coconut coir and peat moss separately, cool them and mix them evenly to prepare a composite amendment; 3) Preparation of improved soil matrix: Mix the composite amendment with the pretreated contaminated soil. The mass ratio of the composite amendment is 25% to 45%. Stir evenly to form improved soil matrix. 4) Addition of functional microorganisms: Add Bacillus subtilis bacterial solution to the improved soil substrate and mix well; 5) Planting: Select healthy seedlings of *Carex balsamina* and plant them in the improved soil obtained in step 4); 6) Maintenance and management: After planting, replenish water regularly to maintain suitable soil moisture. The cultivation period is 100 days. In the middle of the planting period, supplement with Bacillus subtilis inoculum solution and in the later stage of planting, supplement with nitrogen fertilizer.

2. The method for ecological remediation of heavy metal contaminated soil in copper mine tailings ponds based on a composite amendment-Bacillus subtilis-Carex balsamina, as described in claim 1, is characterized in that... In step 2), the drying process involves drying at 105°C for 8 hours.

3. The method for ecological remediation of heavy metal contaminated soil in copper mine tailings ponds based on a composite amendment-Bacillus subtilis-Carex balsamina, as described in claim 1, is characterized in that... In step 2), coconut coir and peat moss are mixed in a mass ratio of 1:

1.

4. The method for ecological remediation of heavy metal contaminated soil in copper mine tailings ponds based on a composite modifier-Bacillus subtilis-Carex balsamina, as described in claim 1, is characterized in that... In step 3), the mass ratio of the composite modifier is 35%.

5. The method for ecological remediation of heavy metal contaminated soil in copper mine tailings ponds based on a composite modifier-Bacillus subtilis-Carex balsamina according to claim 1, characterized in that, In step 4), the effective viable count of the Bacillus subtilis bacterial solution is 1×10⁻⁶. 8 CFU~1×10 9 The concentration of CFU / mL and the addition amount of Bacillus subtilis bacterial solution is 0.2 mL per 0.3 kg of improved soil substrate.

6. The method for ecological remediation of heavy metal contaminated soil in copper mine tailings ponds based on a composite modifier-Bacillus subtilis-Carex balsamina, as described in claim 1, is characterized in that... In step 6), the mid-term after planting is from the 15th to the 30th day after planting, and the late-term after planting is from the 30th to the 50th day after planting.

7. The method for ecological remediation of heavy metal contaminated soil in copper mine tailings ponds based on a composite amendment-Bacillus subtilis-Carex balsamina, as described in claim 1, is characterized in that... In step 6), the effective viable count of the Bacillus subtilis bacterial solution is 1×10⁻⁶. 8 CFU~1×10 9 The concentration of CFU / mL and the addition amount of Bacillus subtilis bacterial solution is 0.2 mL per 0.3 kg of improved soil substrate.

8. The method for ecological remediation of heavy metal contaminated soil in copper mine tailings ponds based on a composite modifier-Bacillus subtilis-Carex balsamina according to claim 1, characterized in that, In step 6), the nitrogen fertilizer is urea.

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