A bacillus velezensis-mediated soil heavy metal passivation bioremediation system

Abstract

This invention discloses a soil heavy metal passivation bioremediation system mediated by Bacillus belyssus, belonging to the field of soil pollution ecological remediation technology. The system is composed of 20-35 parts of domesticated Bacillus belyssus powder, 50-65 parts of organic synergistic carrier, and 10-20 parts of passivation auxiliary agent. The core of this invention utilizes highly stress-resistant Bacillus belyssus acclimated to heavy metal gradients, overcoming the shortcomings of traditional strains such as weak stress resistance and low passivation efficiency. It constructs a triple-coupled remediation mechanism of biological metabolism, physical adsorption, and chemical mineralization, which can efficiently passivate available heavy metals such as lead, cadmium, copper, and zinc in the soil. The passivation rate of composite heavy metals can reach 75%-92%, effectively improving the soil colonization survival rate and metabolic cycle of the strain, simultaneously improving soil aggregate structure, increasing soil organic matter, activating soil enzyme activity, and restoring the soil microecology. This solves the defects of traditional remediation technologies, such as unstable effects, easy secondary activation, and damage to soil fertility.

Description

Technical Field

[0001] This invention belongs to the field of soil heavy metal pollution bioremediation technology, specifically involving a soil heavy metal passivation bioremediation system based on Bacillus belye, which is mediated by a multi-mechanism coupling. It is applicable to in-situ passivation remediation and ecological improvement of soils contaminated with single or multiple heavy metals such as lead, cadmium, copper, and zinc in industrial and mining wastelands, wastewater-irrigated farmland, and areas surrounding industrial parks. It is also applicable to the safe resource utilization of lightly and heavily contaminated farmland, belonging to the interdisciplinary field of environmental remediation and soil ecological improvement. Background Technology

[0002] Soil is the core carrier of agricultural production and ecological environment stability. With the acceleration of my country's industrialization, disorderly mining of mineral resources, illegal discharge of industrial wastewater and waste residue, and long-term excessive application of agricultural fertilizers and pesticides, soil heavy metal pollution has become a core problem restricting the safe use of arable land and ecological environment governance. Heavy metals have the characteristics of non-degradability, bioaccumulation, concealment, and lag, and cannot be eliminated through natural metabolism. Active heavy metals in the soil can migrate with soil moisture and be absorbed by crop roots into the edible parts of crops, accumulating step by step through the food chain, eventually causing health risks such as kidney damage, bone diseases, and heavy metal poisoning in humans, seriously threatening food security and human living ecological security. Moreover, heavy metal stress will destroy the original soil aggregate structure, inhibit the growth and reproduction of native soil microorganisms, reduce soil enzyme activity, lead to slow decomposition of soil organic matter and obstruction of nutrient cycling, resulting in soil fertility decline and arable land quality degradation.

[0003] Currently, domestic and international remediation technologies for heavy metal pollution in soil are mainly divided into three categories: physical remediation, chemical remediation, and bioremediation. Among them, bioremediation technology, with its core advantages of being green and pollution-free, being applied in situ, being eco-friendly, and being able to improve soil, has become the mainstream technology to replace traditional physical and chemical remediation. Microbial passivation remediation is a research hotspot. Currently, most existing remediation strains are conventional strains such as Bacillus subtilis, Bacillus licheniformis, Pseudomonas, and Lactobacillus, which have obvious technical shortcomings: First, they have low heavy metal tolerance thresholds, and under high concentrations of heavy metal stress, these strains are easily inactivated and lysed, making them unsuitable for remediating heavily polluted soils. Second, they have weak metabolic activity, limited ability to produce acid and extracellular polysaccharides, low passivation efficiency for heavy metals, and poor compatibility with complex heavy metals. Third, they have weak soil colonization capacity, with significant competitive inhibition from native microorganisms, and exogenous strains rapidly decay after being applied to the soil, unable to maintain remediation activity in the long term. Fourth, existing microbial remediation systems have simple formulations, relying only on the action of a single strain, lacking carrier survival and synergistic passivation design with adjuvants, resulting in limited remediation functions and an inability to simultaneously achieve heavy metal passivation and soil ecological restoration.

[0004] Bacillus velezensis is a novel probiotic strain discovered in recent years. A Gram-positive bacterium, it possesses strong environmental resistance, metabolic activity, and ecological compatibility. It stably secretes small-molecule organic acids such as citric acid, malic acid, and succinic acid, as well as active metabolites such as extracellular polysaccharides, mucoproteins, and antimicrobial peptides. Existing research confirms that its metabolites can complex, precipitate, and mineralize with heavy metal ions in soil, converting highly reactive ionic heavy metals into low-reactivity carbonates, phosphates, and hydroxides. Simultaneously, it can optimize soil microbial community structure and enhance soil enzyme activity. However, the application of Bacillus velezensis in soil remediation is still in the laboratory research stage. A standardized, systematic, and engineered remediation system has not yet been established. Common problems include unacclimated strains, low inoculant activity, unscientific formulation ratios, lack of synergistic passivation systems, and non-standard construction techniques. These issues result in low soil colonization rates, unstable passivation effects, and inability to adapt to complex site pollution remediation, significantly limiting the industrial application of this strain in the field of heavy metal remediation. Summary of the Invention

[0005] To address the shortcomings of existing soil heavy metal remediation technologies, such as low passivation efficiency, poor stability, easy secondary activation, damage to soil ecology, weak adaptability to complex pollution, and inability to simultaneously improve soil quality and fertility, this invention provides a soil heavy metal passivation bioremediation system mediated by Bacillus belyssus. Using highly stress-resistant Bacillus belyssus, specifically domesticated for heavy metals, as the core, combined with organic survival-enhancing carriers and multifunctional passivation adjuvants, a triple-coupled remediation mechanism of biological metabolic passivation, physical adsorption and fixation, and chemical complexation mineralization is constructed. This achieves efficient, stable, and long-lasting passivation of lead, cadmium, copper, and zinc complex heavy metals, while simultaneously restoring soil microecology, improving soil structure, and enhancing soil fertility. This integrated engineering application achieves heavy metal detoxification, ecological restoration, and farmland quality improvement in polluted soil.

[0006] To achieve the above-mentioned objectives, this invention provides a soil heavy metal passivation bioremediation system mediated by Bacillus vesiculosus, comprising a composite functional bacterial agent, an organic synergistic carrier, and a passivation auxiliary agent. The composite functional bacterial agent is a highly active solid powder of Bacillus vesiculosus acclimated to heavy metal gradients, which is the core functional component for achieving biopassivation. The organic synergistic carrier is a compound system of well-rotted organic fertilizer, humic acid, and straw charcoal, used to maintain the bacterial strain, improve the soil, and provide a metabolic substrate. The passivation auxiliary agent is a compound system of citric acid-modified potassium dihydrogen phosphate, nano-diatomaceous earth, and chelated amino acids, used to synergistically enhance the heavy metal passivation effect and improve the remediation stability. The components of the remediation system are precisely proportioned by mass: 20-35 parts of Bacillus vesiculosus powder, 50-65 parts of organic synergistic carrier, and 10-20 parts of passivation auxiliary agent. The components are synergistically combined without antagonistic effects, maximizing the coupled remediation effect.

[0007] Furthermore, the *Bacillus vesiculosus* strain is an artificially acclimatized strain with gradient heavy metals. After being acclimatized and cultured under combined stress of cadmium, lead, copper, and zinc, it possesses extremely high tolerance to heavy metals. The effective viable count of the *Bacillus vesiculosus* powder is ≥20 billion CFU / g, the contamination rate is ≤0.5%, and the moisture content is ≤8%. The strain's tolerance range for heavy metal concentrations is: cadmium 0–120 mg / kg, lead 0–800 mg / kg, copper 0–500 mg / kg, and zinc 0–1000 mg / kg, making it suitable for the remediation of moderately to severely contaminated soils with multiple heavy metals.

[0008] Furthermore, the standardized preparation method of the Bacillus vesiculosus powder includes the following steps: (1) Activation of strain: The low-temperature preserved Bacillus belye strain was inoculated into solid LB medium and cultured at 32℃ inverted for 24h to obtain single colonies. The dominant single colonies were selected for slant subculturing twice to ensure the stability of the strain activity. (2) Heavy metal gradient acclimatization: The activated strains were inoculated into LB liquid medium containing gradient complex heavy metals (cadmium 5-120 mg / kg, lead 50-800 mg / kg), and acclimatized at 32℃ and 200 r / min with shaking. The heavy metal concentration was gradually increased and acclimatized for 5 generations to screen out the dominant strains with high heavy metal tolerance. (3) Liquid fermentation culture: The domesticated dominant strain was inoculated into the liquid fermentation medium at an inoculation rate of 5%. The medium formula was: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 8 g / L glucose, and natural pH. The culture conditions were 30-35℃, 180-220 r / min shaking culture for 24-36 h to obtain a fermentation stock solution with a viable count ≥8 billion CFU / mL. (4) Preparation of bacterial powder: The fermentation liquid and sterile diatomaceous earth are mixed evenly at a volume-to-mass ratio of 3:1. The low-temperature vacuum drying process at -40℃ is adopted, the vacuum degree is controlled at 0.08~0.09MPa, and the drying time is 12~16h. After drying, the material is crushed through an 80-mesh standard sieve, sealed and stored in the dark to obtain highly active Bacillus vesiculosus bacterial powder.

[0009] Furthermore, the organic enhancement carrier, by mass fraction, comprises: 30-40 parts of well-rotted livestock and poultry organic fertilizer, 10-15 parts of humic acid, and 10-15 parts of straw charcoal. The parameters of each component are strictly defined: the well-rotted livestock and poultry organic fertilizer is a high-temperature aerobic composting product with a composting degree ≥90%, an organic matter content ≥45%, free of insect eggs, pathogens, and weed seeds, and rich in readily available nutrients; the humic acid is a high-purity mineral-derived humic acid with a purity ≥95%, possessing excellent complexing, adsorption, and water and fertilizer retention capabilities; the straw charcoal is a high-temperature anaerobic carbonization product of corn straw at 500-600℃, with a pore size of 20-50 nm, a specific surface area ≥180 m² / g, and a well-developed pore structure, which can serve as a colonization carrier for bacterial strains and a heavy metal adsorption framework.

[0010] Furthermore, the passivation auxiliary agent, by mass parts, comprises: 4-8 parts modified potassium dihydrogen phosphate, 3-6 parts nano-diatomaceous earth, and 3-6 parts chelated amino acids; the parameters of each component are strictly limited: the modified potassium dihydrogen phosphate is a citric acid surface-modified product with a water-soluble phosphorus content ≥52%, which can slowly release phosphate ions and form stable phosphate precipitates with heavy metals; the nano-diatomaceous earth has a particle size of 20-50 nm, a porosity ≥85%, a large specific surface area, and extremely strong physical adsorption capacity; the chelated amino acids are a complex plant-derived amino acid chelate prepared by fermentation of soybeans and corn, with a total amino acid content ≥30%, which can chelate with heavy metal ions and reduce the biotoxicity of heavy metals.

[0011] Furthermore, the overall preparation process of the repair system is as follows: accurately weigh the organic synergist and passivation auxiliary agent according to the ratio, put them into a horizontal mixer, and stir at low speed for 10 minutes to achieve premixing; then add Bacillus vesiculosus powder, stir at low speed for 15 minutes, and operate at room temperature, away from light, and in a closed environment throughout the process to avoid inactivation of the strain. After mixing evenly, seal and package, and store at room temperature in a cool place to obtain the finished repair system.

[0012] Furthermore, the in-situ repair process includes the following standardized steps: S1. Soil pretreatment: Remove stones, weeds, plastic debris and other waste from the soil surface manually or mechanically. Use a deep tiller to plow the soil to a depth of 20-30cm, break up soil clods and level the land. Adjust the soil moisture content to 18%-25% by sprinkling water or drying to ensure that the soil is loose and breathable, providing good conditions for bacterial colonization and material mixing. S2. Application of the remediation system: Adjust the application amount according to the degree of soil pollution. Apply 80-100 kg per acre for lightly polluted soil and 100-120 kg per acre for moderately to heavily polluted soil. Apply manually or by mechanized application with a fertilizer spreader to ensure uniform distribution of materials in the surface soil. S3. Soil Mixing and Curing: Deeply till the soil again to a depth of 15-25cm to fully mix the remediation system with the 0-30cm topsoil layer, eliminating material stratification; use mulch film for full coverage and moisture retention for 20-30 days, strictly controlling the soil moisture content to 20%-28% and the ambient temperature to 15-35℃ during the curing period, avoiding strong sunlight exposure and soil drought, to provide a stable environment for the reproduction and metabolism of Bacillus belye. S4. Post-maintenance and reuse: After the maintenance period ends, remove the mulch film and allow it to air dry naturally for 3-5 days. Once the soil moisture content has dropped to the normal level in the field, crop planting or site restoration can be carried out.

[0013] Compared with existing traditional chemical passivation and common microbial remediation technologies, this invention has the following outstanding advantages: 1. This invention uses Bacillus belye, which has been domesticated with gradient heavy metals, to stably survive and reproduce under high concentrations of compound heavy metal stress. The strain continuously secretes small molecule organic acids, extracellular polysaccharides, and functional proteins. Through multiple biological mechanisms such as complexation, adsorption, biomineralization, and redox, it targets and passivates available lead, cadmium, copper, and zinc in the soil. The overall passivation rate of compound heavy metals can reach 75% to 92%, which is far higher than that of conventional microbial remediation technologies. It can be adapted to soils contaminated with single or compound heavy metals at the same time, solving the technical problems of poor adaptability, inactivation in highly polluted environments, and low passivation efficiency of traditional strains.

[0014] 2. This invention constructs a triple-coupled remediation system of biological metabolic passivation, carbon-based physical adsorption, and chemical complexation mineralization. It physically locks heavy metals through the well-developed porous structure of straw charcoal and nano-diatomite, continuously releases phosphate ions from modified phosphates to form stable, insoluble phosphate precipitates with heavy metals, and achieves deep complexation and fixation with strain metabolites and amino acid chelates. The multiple fixation mechanisms work synergistically and complement each other to enhance the effect, which solves the defects of traditional passivation technology, such as reversible effects and easy secondary activation by rainwater leaching. The stability of heavy metals after remediation is greatly improved, and the long-term remediation period can reach more than 3 years.

[0015] 3. The organic enhancement carrier of this invention can provide a continuous carbon source, nitrogen source and colonization site for the strains of this invention, greatly improving the soil colonization rate and survival period of the strains. The colonization period of the strains can reach more than 60 days. At the same time, it can significantly increase the soil organic matter content, optimize the soil aggregate structure, reduce the soil bulk density, increase the soil porosity, activate the activity of soil urease, phosphatase and sucrase, repair the damaged soil micro-ecology, improve the diversity of soil microbial community, and achieve the simultaneous completion of heavy metal detoxification, soil fertility improvement and ecological restoration, which is suitable for the sustainable and safe production needs of arable land.

[0016] 4. All components of this invention are natural, environmentally friendly, and biodegradable ecological raw materials. There are no chemically synthesized toxic components. After being applied to the soil, there are no residues or secondary pollution. It will not cause secondary problems such as soil compaction or salinization. The remediation process is simple, and the entire process is carried out in situ. There is no need for large-scale precision equipment or soil transportation from other places. The construction cost is reduced by 40% to 60% compared with traditional physical and chemical remediation. It can be adapted to various pollution scenarios such as farmland, mining areas, industrial sites, and garden greening areas. It has extremely high value for large-scale promotion.

[0017] 5. The remediation system of this invention has been optimized for stress resistance and can stably exert its remediation effect in a wide temperature range of 15-35℃, in slightly saline-alkali soils, and in soils of different textures. It is not strictly limited by regional climate or soil texture and its environmental adaptability far exceeds that of conventional remediation microbial agents. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0019] Description of core raw materials and equipment for this invention: Core strain: Bacillus belye, acclimatized under a gradient of combined cadmium, lead, copper, and zinc stress. The strain tolerates the following concentration ranges: cadmium 0–120 mg / kg, lead 0–800 mg / kg, copper 0–500 mg / kg, and zinc 0–1000 mg / kg. The effective viable count of the bacterial powder is ≥20 billion CFU / g, the contamination rate is ≤0.5%, and the moisture content is ≤8%.

[0020] Organic enhancement carriers: well-rotted livestock and poultry organic fertilizer (high-temperature aerobic composting, composting degree ≥90%, organic matter content ≥45%, free of insect eggs, pathogens, and weed seeds), mineral-derived high-purity humic acid (purity ≥95%, with excellent complexing, adsorption, water and fertilizer retention capabilities), and corn straw charcoal (500~600℃ anaerobic high-temperature carbonization, pore size 20~50nm, specific surface area ≥180m² / g, and well-developed pore structure).

[0021] Passivation aids: Citric acid modified potassium dihydrogen phosphate (water-soluble phosphorus content ≥52%, can slowly release phosphate ions), nano diatomaceous earth (particle size 20-50nm, porosity ≥85%, extremely strong physical adsorption capacity), plant-derived complex amino acid chelate (prepared by fermentation of soybeans and corn, total amino acid content ≥30%, can chelate with heavy metal ions).

[0022] Main equipment: horizontal mixing machine, deep tillage machine, agricultural mulch film, LB solid / liquid culture medium, constant temperature shaking incubator, -40℃ low temperature vacuum drying oven, 80 mesh standard test sieve, soil heavy metal test kit, soil fertility test kit.

[0023] Example 1 (Remediation of farmland with moderate lead-cadmium combined pollution): This embodiment provides a soil heavy metal passivation bioremediation system mediated by Bacillus belyssus, comprising a compound functional microbial agent, an organic synergistic carrier, and a passivation auxiliary agent. The compound functional microbial agent is a highly active solid powder of Bacillus belyssus acclimated to heavy metal gradients. The organic synergistic carrier is a compound system of well-rotted organic fertilizer, humic acid, and straw charcoal. The passivation auxiliary agent is a compound system of citric acid-modified potassium dihydrogen phosphate, nano-diatomaceous earth, and chelated amino acids.

[0024] The components of the repair system, by weight, are: 25 parts of Bacillus vesiculosus powder, 60 parts of organic synergistic carrier, and 15 parts of passivation auxiliary agent.

[0025] The organic enhancement carrier, by weight, consists of: 35 parts of well-rotted livestock and poultry organic fertilizer, 12 parts of humic acid, and 13 parts of straw charcoal; the passivation auxiliary agent, by weight, consists of: 6 parts of modified potassium dihydrogen phosphate, 5 parts of nano-diatomite, and 4 parts of chelated amino acids; and the effective viable count of Bacillus vesiculosus powder is ≥20 billion CFU / g, after being acclimatized by a gradient of lead and cadmium combined stress.

[0026] The preparation methods of Bacillus vesiculosus powder include: (1) Activation of strain: The low-temperature preserved Bacillus belye strain was inoculated into solid LB medium and cultured at 32℃ inverted for 24h to obtain single colonies. The dominant single colonies were selected for slant subculturing twice to ensure the stability of the strain activity. (2) Heavy metal gradient acclimatization: The activated strains were inoculated into LB liquid medium containing gradient compound heavy metals (cadmium 10 mg / kg, lead 85 mg / kg) and cultured under shaking acclimatization at 32℃ and 200 r / min. The heavy metal concentration was gradually increased and acclimatized for 5 generations to screen for high heavy metal tolerance dominant strains. (3) Liquid fermentation culture: The domesticated dominant strain was inoculated into the liquid fermentation medium at an inoculation rate of 5%. The medium formula was: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 8 g / L glucose, and natural pH. The culture conditions were 32℃, 200 r / min shaking culture for 30 h to obtain a fermentation stock solution with a viable count ≥8 billion CFU / mL. (4) Preparation of bacterial powder: The fermentation liquid and sterile diatomaceous earth were mixed evenly at a volume-to-mass ratio of 3:1. The mixture was dried at -40℃ using a low-temperature vacuum drying process with the vacuum degree controlled at 0.085MPa and the drying time being 14h. After drying, the material was crushed through an 80-mesh standard sieve, sealed and stored away from light to obtain highly active Bacillus vesiculosus bacterial powder.

[0027] The preparation process of the remediation system is as follows: accurately weigh the organic synergist and passivation auxiliary agent according to the formula, and put them into a horizontal mixer. First, mix the organic synergist and passivation auxiliary agent and stir at low speed for 10 minutes to achieve premixing. Then add Bacillus vesiculosus powder and stir at low speed for 15 minutes. The entire process is carried out at room temperature, away from light, and in a closed environment to avoid inactivation of the strain. After mixing evenly, seal and package, and store at room temperature in a cool place to obtain the finished remediation system.

[0028] Test site: A wastewater-irrigated farmland in southern China. The soil texture is loam. The soil test indicators in the 0-30cm topsoil layer are: available cadmium 1.85mg / kg, available lead 82.6mg / kg, and soil organic matter 18.2g / kg. The soil is relatively infertile and has low microbial activity, belonging to farmland with moderate lead and cadmium combined pollution.

[0029] In-situ restoration technology includes the following steps: Step S1, Soil pretreatment: Remove stones, weeds, plastic debris and other waste from the soil surface manually or mechanically, and use a deep tiller to plow the soil to a depth of 25cm, break up soil clods and level the land. Adjust the soil moisture content to 22% by watering or sun drying to ensure that the soil is loose and breathable, providing good conditions for bacterial colonization and material mixing. Step S2, application of the remediation system: Adjust the application amount according to the degree of soil pollution. Apply 85 kg of the remediation system per acre for lightly polluted soil, and apply 110 kg per acre for moderately to heavily polluted soil. Apply the system manually or by mechanized application with a fertilizer spreader to ensure uniform distribution of the material in the surface soil. Step S3, Soil Mixing and Curing: Deeply till the soil again to a depth of 20cm to thoroughly mix the remediation system with the 0-30cm topsoil layer, eliminating material stratification; use mulch film for full coverage and moisture retention for 25 days, maintaining a stable soil moisture content of 22%-25% and an ambient temperature of 20-30℃ during the curing period; avoid strong sunlight exposure and soil drought to provide a stable environment for the reproduction and metabolism of Bacillus belyssus. Step S4, Post-maintenance and reuse: After the maintenance period ends, remove the mulch film and allow it to air dry naturally for 4 days. Once the soil moisture content has dropped to the normal level in the field, crop planting or site restoration can be carried out.

[0030] Remediation effect testing: Samples were taken 7 days after remediation was completed and tested. The available cadmium in the soil decreased to 0.28 mg / kg, with a passivation rate of 84.8%; the available lead decreased to 9.8 mg / kg, with a passivation rate of 88.1%; the soil organic matter increased to 21.6 g / kg, an increase of 18.6%; the activities of soil urease and phosphatase increased by 2.1 times and 1.8 times respectively; the number of soil microorganisms increased by 2.3 times; and the soil looseness was significantly improved, fully meeting the requirements for safe planting of crops.

[0031] Example 2 (Remediation of a heavily contaminated industrial site with combined copper and zinc pollution): This embodiment provides a soil heavy metal passivation bioremediation system mediated by Bacillus vesiculus, comprising a compound functional microbial agent, an organic synergistic carrier, and a passivation auxiliary agent. The compound functional microbial agent is a highly active solid powder of Bacillus vesiculus mediated by heavy metal gradient acclimatization. The organic synergistic carrier is a compound system of well-rotted organic fertilizer, humic acid, and straw charcoal. The passivation auxiliary agent is a compound system of citric acid-modified potassium dihydrogen phosphate, nano-diatomaceous earth, and chelated amino acids. The components of the remediation system, by mass, are: 30 parts of Bacillus vesiculus molecule powder, 55 parts of organic synergistic carrier, and 15 parts of passivation auxiliary agent.

[0032] The organic enhancement carrier, by weight, consists of: 32 parts of well-rotted livestock and poultry organic fertilizer, 13 parts of humic acid, and 10 parts of straw charcoal; the passivation auxiliary agent, by weight, consists of: 7 parts of modified potassium dihydrogen phosphate, 4 parts of nano-diatomaceous earth, and 4 parts of chelated amino acids; and the effective viable count of Bacillus vesiculosus powder is ≥20 billion CFU / g, after being acclimatized by a copper and zinc composite stress gradient.

[0033] The preparation methods of Bacillus vesiculosus powder include: (1) Activation of strain: The low-temperature preserved Bacillus belye strain was inoculated into solid LB medium and cultured at 32℃ inverted for 24h to obtain single colonies. The dominant single colonies were selected for slant subculturing twice to ensure the stability of the strain activity. (2) Heavy metal gradient acclimatization: The activated strains were inoculated into LB liquid medium containing gradient compound heavy metals (copper 125 mg / kg, zinc 400 mg / kg) and cultured under shaking acclimatization at 32℃ and 200 r / min. The heavy metal concentration was gradually increased and acclimatized for 5 generations to screen out the dominant strains with high heavy metal tolerance. (3) Liquid fermentation culture: The domesticated dominant strain was inoculated into the liquid fermentation medium at an inoculation rate of 5%. The medium formula was: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 8 g / L glucose, and natural pH. The culture conditions were 33℃, 210 r / min shaking culture for 32 h to obtain a fermentation stock solution with a viable count ≥8 billion CFU / mL. (4) Preparation of bacterial powder: The fermentation liquid and sterile diatomaceous earth were mixed evenly at a volume-to-mass ratio of 3:1. The mixture was dried at -40℃ in a low-temperature vacuum process with the vacuum degree controlled at 0.087MPa for 15 hours. After drying, the material was crushed and passed through an 80-mesh standard sieve. It was then sealed and stored away from light to obtain highly active Bacillus vesiculosus bacterial powder.

[0034] The preparation process of the remediation system is as follows: accurately weigh the organic synergist and passivation auxiliary agent according to the formula, and put them into a horizontal mixer. First, mix the organic synergist and passivation auxiliary agent and stir at low speed for 10 minutes to achieve premixing. Then add Bacillus vesiculosus powder and stir at low speed for 15 minutes. The entire process is carried out at room temperature, away from light, and in a closed environment to avoid inactivation of the strain. After mixing evenly, seal and package, and store at room temperature in a cool place to obtain the finished remediation system.

[0035] Test site: An abandoned electroplating industrial site. The soil is clay. The available copper content in the 0-30cm soil is 126.3mg / kg, the available zinc content is 385.7mg / kg, and the soil organic matter content is 16.7g / kg. The heavy metal pollution level is high, and it belongs to a heavily copper-zinc combined pollution industrial site. The soil is severely compacted, lacks organic matter, and has a single microbial community.

[0036] In-situ restoration technology includes the following steps: Step S1, Soil pretreatment: Remove stones, weeds, plastic debris and other waste from the soil surface manually or mechanically, and use a deep tiller to plow the soil to a depth of 30cm, break up soil clods and level the land. Adjust the soil moisture content to 24% by watering or sun drying to ensure that the soil is loose and breathable, providing good conditions for bacterial colonization and material mixing. Step S2, Application of the remediation system: Adjust the application amount according to the degree of soil pollution. For lightly polluted soil, apply 100 kg of the remediation system per acre in this embodiment. For heavily polluted soil, apply 120 kg per acre. Apply manually or by mechanized application with a fertilizer spreader to ensure uniform distribution of materials in the surface soil. Step S3, Soil Mixing and Curing: Deeply till the soil again to a depth of 25cm to thoroughly mix the remediation system with the 0-30cm topsoil layer, eliminating material stratification; use mulch film for full coverage and moisture retention for 28 days, maintaining a stable soil moisture content of 22%-27% and an ambient temperature of 23-26℃ during the curing period; avoid strong sunlight exposure and soil drought to provide a stable environment for the reproduction and metabolism of Bacillus belyssus. Step S4, Post-maintenance and reuse: After the maintenance period ends, remove the mulch film and allow it to air dry naturally for 5 days. Once the soil moisture content has dropped to the normal level in the field, crop planting or site restoration can be carried out.

[0037] Remediation effect testing: Samples were taken and tested 7 days after remediation. The available copper in the soil decreased to 17.5 mg / kg, with a passivation rate of 86.1%; the available zinc decreased to 40.5 mg / kg, with a passivation rate of 89.5%; the soil organic matter increased to 19.6 g / kg, an increase of 17.8%; soil compaction was completely improved; soil porosity increased by 25.3%; soil microbial community diversity index increased by 38.7%; the site's soil ecological function was basically restored, meeting the standards for site revegetation and secondary development.

[0038] Example 3 (Remediation of farmland with mild multi-component heavy metal pollution): This embodiment provides a soil heavy metal passivation bioremediation system mediated by Bacillus vesiculus, comprising a composite functional microbial agent, an organic synergistic carrier, and a passivation auxiliary agent. The composite functional microbial agent is a highly active solid powder of Bacillus vesiculus mediated by heavy metal gradient acclimation. The organic synergistic carrier is a compound system of well-rotted organic fertilizer, humic acid, and straw charcoal. The passivation auxiliary agent is a compound system of citric acid-modified potassium dihydrogen phosphate, nano-diatomaceous earth, and chelated amino acids. The components of the remediation system, by mass, are: 20 parts of Bacillus vesiculus 20 parts, 65 parts of organic synergistic carrier, and 15 parts of passivation auxiliary agent.

[0039] The organic enhancement carrier, by weight, consists of: 35 parts of well-rotted livestock and poultry organic fertilizer, 12 parts of humic acid, and 13 parts of straw charcoal; the passivation auxiliary agent, by weight, consists of: 6 parts of modified potassium dihydrogen phosphate, 5 parts of nano-diatomite, and 4 parts of chelated amino acids; and the effective viable count of Bacillus vesiculosus powder is ≥20 billion CFU / g, after being acclimatized by a gradient of lead and cadmium combined stress.

[0040] The preparation methods of Bacillus vesiculosus powder include: (1) Activation of strain: The low-temperature preserved Bacillus belye strain was inoculated into solid LB medium and cultured at 32℃ inverted for 24h to obtain single colonies. The dominant single colonies were selected for slant subculturing twice to ensure the stability of the strain activity. (2) Heavy metal gradient acclimatization: The activated strains were inoculated into LB liquid medium containing gradient complex heavy metals (copper 110 mg / kg, zinc 325 mg / kg, cadmium 15 mg / kg, lead 165 mg / kg) and cultured under shaking acclimatization at 32℃ and 200 r / min. The heavy metal concentration was gradually increased and acclimatized for 5 generations to screen out the dominant strains with high heavy metal tolerance. (3) Liquid fermentation culture: The domesticated dominant strain was inoculated into the liquid fermentation medium at an inoculation rate of 5%. The medium formula was: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 8 g / L glucose, and natural pH. The culture conditions were 30℃ and 180 r / min shaking culture for 30 h to obtain a fermentation stock solution with a viable count ≥8 billion CFU / mL. (4) Preparation of bacterial powder: The fermentation liquid and sterile diatomaceous earth were mixed evenly at a volume-to-mass ratio of 3:1. The mixture was dried at -40℃ under vacuum, with the vacuum degree controlled at 0.082MPa and the drying time at 13h. After drying, the material was crushed through an 80-mesh standard sieve, sealed and stored away from light to obtain highly active Bacillus vesiculosus bacterial powder.

[0041] The preparation process of the remediation system is as follows: accurately weigh the organic synergist and passivation auxiliary agent according to the formula, and put them into a horizontal mixer. First, mix the organic synergist and passivation auxiliary agent and stir at low speed for 10 minutes to achieve premixing. Then add Bacillus vesiculosus powder and stir at low speed for 15 minutes. The entire process is carried out at room temperature, away from light, and in a closed environment to avoid inactivation of the strain. After mixing evenly, seal and package, and store at room temperature in a cool place to obtain the finished remediation system.

[0042] Test site: A wastewater-irrigated farmland in southern China. The soil texture is loam. The soil test indicators in the 0-30cm topsoil layer are: available copper 103.75mg / kg, available zinc 303.75mg / kg, available cadmium 13.75mg / kg, available lead 152.6mg / kg, and soil organic matter 18.2g / kg. The soil is relatively infertile and has low microbial activity, belonging to farmland with mild multiple complex heavy metal pollution.

[0043] In-situ restoration technology includes the following steps: Step S1, Soil pretreatment: Remove stones, weeds, plastic debris and other waste from the soil surface manually or mechanically, and use a deep tiller to plow the soil to a depth of 25cm, break up soil clods and level the land. Adjust the soil moisture content to 22% by watering or sun drying to ensure that the soil is loose and breathable, providing good conditions for bacterial colonization and material mixing. Step S2, application of the remediation system: Adjust the application amount according to the degree of soil pollution. For lightly polluted farmland, apply 80-90 kg per mu. In this embodiment, 85 kg of the remediation system is applied. For moderately to heavily polluted soil, apply 110 kg per mu. Apply manually or by mechanized application with a fertilizer spreader to ensure uniform distribution of materials in the surface soil. Step S3, Soil Mixing and Curing: Deeply till the soil again to a depth of 20cm to fully mix the remediation system with the 0-30cm topsoil layer, eliminating material stratification; use mulch film for full coverage and moisture retention for 25 days, during which the soil moisture content is maintained at a stable 22%-25%, the ambient temperature is 20-30℃, and strong sunlight exposure and soil drought are avoided to provide a stable environment for the reproduction and metabolism of Bacillus belyssus. Step S4, Post-maintenance and reuse: After the maintenance period ends, remove the mulch film and allow it to air dry naturally for 4 days. Once the soil moisture content has dropped to the normal level in the field, crop planting or site restoration can be carried out.

[0044] Remediation effect testing: Samples were taken 7 days after remediation was completed and tested. The passivation rate of available lead, cadmium, copper and zinc in the soil was ≥75%, the soil organic matter increased by more than 15%, and the soil fertility was significantly improved, which can realize the safe and efficient resource utilization of lightly polluted farmland.

[0045] Comparative Example 1 (Traditional Chemical Passivation Repair): The same contaminated farmland soil as in Example 1 was selected and remediated using a commercially available composite chemical passivating agent of lime and bentonite, with the same construction process and maintenance cycle.

[0046] Post-repair testing revealed that the cadmium passivation rate was 62.3% and the lead passivation rate was 65.7%, with passivation effects far lower than those of this invention. Simultaneously, the soil pH value increased from 6.8 to 8.3, indicating mild salinization, and soil compaction worsened. Soil microbial activity decreased by 27.4%, and soil organic matter did not improve. Only limited heavy metal fixation was achieved, and the soil ecology could not be improved.

[0047] Comparative Example 2 (Remediation with a single unacclimated Bacillus belye inoculum): The same contaminated soil as in Example 1 was selected and treated separately with ordinary Bacillus vesiculosus powder of the same number of viable bacteria and without heavy metal acclimation, without organic carriers or passivation agents, under the same construction conditions.

[0048] Post-remediation testing revealed that the cadmium passivation rate was 71.2% and the lead passivation rate was 73.5%, with passivation effects significantly lower than those of the present invention. Furthermore, the strain had poor soil colonization ability, and the survival rate of the strain was less than 30% after maintenance. The remediation effect showed poor stability, and a small amount of heavy metal activation was detected after 30 days, while there was no soil improvement effect.

[0049] Comparative Example 3 (Conventional Microbial Composite Remediation System): The conventional remediation system of common Bacillus and humic acid disclosed in existing patents was used to remediate the soil of Example 1 under the same process conditions.

[0050] Post-remediation testing revealed that the overall passivation rate of heavy metals was only about 68%, the strain exhibited poor stress resistance, and its activity rapidly declined under high temperature and drought conditions, resulting in insufficient long-term remediation capacity and weak soil improvement effect. Comparison of soil improvement effects between Examples 1-3 and Comparative Examples 1-3: Example 1 (Remediation of Moderate Lead and Cadmium Contamination) Domestication of Bacillus belye, organic synergist, and passivation aid Cadmium 84.8%, Lead 88.1% 18.60% Urease levels increased 2.1 times, phosphatase levels increased 1.8 times, and the number of microorganisms increased 2.3 times. Soil looseness significantly improved Long-term repair cycle ≥ 3 years, no risk of secondary activation No secondary pollution, no salinization or hardening problems Example 2 (Remediation of Severe Copper-Zinc Contamination) Domestication of Bacillus belye, organic synergist, and passivation aid Copper 86.2%, Zinc 89.5% 17.80% The microbial community diversity index increased by 38.7%. Soil porosity increased by 25.3%, and soil compaction was completely improved. Long-term repair cycle ≥ 3 years, no risk of secondary activation No secondary pollution, no salinization or hardening problems Example 3 (Remediation of Mild Complex Pollution) Domestication of Bacillus belye, organic synergist, and passivation aid Lead, cadmium, copper, and zinc all ≥75% ≥15% Soil enzyme activity significantly increased Soil aggregate structure optimization Long-term repair cycle ≥ 3 years, no risk of secondary activation No secondary pollution, no salinization or hardening problems Comparative Example 1 (Traditional Chemical Passivation) Lime and bentonite composite passivating agent Cadmium 62.3%, Lead 65.7% No improvement Microbial activity decreased by 27.4%. Soil compaction worsens The effect is reversible; rainwater leaching easily leads to secondary activation. Mild salinization and increased soil compaction Comparative Example 2 (Unacclimated single microbial agent) Undomesticated Bacillus belye powder Cadmium 71.2%, Lead 73.5% No improvement No improvement No improvement The survival rate of the strain was <30%, and heavy metal activation occurred after 30 days. No secondary pollution, but no soil improvement effect. Comparative Example 3 (Conventional Microbial Complex System) Common Bacillus, humic acid The overall passivation rate is approximately 68%. Slight improvement slight improvement Slight improvement The strain exhibits poor stress resistance, rapid activity decay, and insufficient long-lasting effect. No secondary pollution, but the remediation effect is unstable. In summary, based on the comparison of soil improvement effects between Examples 1-3 and Comparative Examples 1-3, it can be concluded that: 1. The heavy metal passivation ability of the domesticated Bacillus belye composite remediation system of this invention is far superior to that of traditional remediation methods: The three implementation examples target single, combined, and moderately to heavily contaminated soils with lead / cadmium / copper / zinc pollution, achieving a passivation rate of up to 75% and up to 89.5% for the available heavy metals.

[0051] Traditional chemical passivation (Comparative Example 1) had a passivation rate of only 62.3%~65.7%, an undomesticated single strain (Comparative Example 2) had a passivation rate of 71.2%~73.5%, and an ordinary microbial system (Comparative Example 3) had an overall passivation rate of only 68%.

[0052] After being domesticated with gradient heavy metals, Bacillus belye, combined with an organic carrier and a passivation aid, forms a triple coupling mechanism to fix heavy metals through multiple pathways. Its passivation efficiency is significantly higher than that of existing technologies, making it suitable for various complex heavy metal pollution scenarios in farmland and industrial sites.

[0053] 2. This invention can simultaneously improve soil fertility and microecology: This invention, along with its organic enhancement carrier, increases soil organic matter by 15% to 18.6% after remediation, significantly improves soil urease and phosphatase activity, total microbial count, and community diversity, optimizes soil aggregate structure, increases porosity, and thoroughly improves soil compaction.

[0054] Comparative Example 1: Lime chemical passivation causes soil salinization, increased compaction, and a 27.4% decrease in microbial activity, damaging the basic fertility of arable land. Comparative Example 2: Single microbial agent and Comparative Example 3: Ordinary microbial system can hardly improve organic matter, have a very weak effect on improving soil enzymes, microorganisms, and soil structure, and can only fix heavy metals without the function of fertilization and soil improvement.

[0055] This invention offers strong and long-lasting repair effects, with no risk of secondary activation by heavy metals. This invention employs a triple fixation mechanism of biological metabolism, physical adsorption, and chemical mineralization, resulting in stable heavy metal binding morphology and a long-term repair cycle of over 3 years. After maintenance, there will be no reactivation of heavy metals.

[0056] Comparative Example 1: Chemical passivation relies on acid-base precipitation. Heavy metals are easily released again after being dissolved by rainwater, and the remediation effect is reversible. Comparative Example 2: The strain was not domesticated and had no survival carrier. The survival rate of soil colonization was less than 30%. Heavy metal activation occurred 30 days after short-term remediation. Comparative Example 3: Common Bacillus has poor resistance to adversity. The strain is rapidly inactivated under high temperature and drought conditions, and the long-term remediation effect is significantly reduced.

[0057] 4. This invention has high environmental safety, causes no secondary soil pollution, and is suitable for long-term safe production on arable land: All raw materials are natural and biodegradable materials such as organic fertilizer, straw charcoal, amino acids, and modified phosphates. After application, it will not cause secondary soil problems such as salinization and compaction, and can be directly used for planting crops on farmland.

[0058] Traditional lime passivating agents can significantly increase soil pH, disrupting the acid-base balance of farmland and making them unsuitable for long-term cultivation. While single-agent microbial agents and ordinary microbial systems do not involve chemical pollution, they lack soil conditioning components and cannot improve the quality of arable land.

[0059] 5. The synergistic effect of the formulation is irreplaceable; the domesticated strains, organic carriers, and passivation agents are all indispensable. The passivation effect and colonization ability of the strain using only unacclimated strains (Comparative Example 2) declined significantly, proving that heavy metal gradient acclimation is the core of the strain's high stress resistance and high passivation activity. When organic carriers and passivation aids are lacking, the strains cannot be provided with a carbon source for colonization, and there are also no synergistic components of physical adsorption and chemical mineralization, resulting in a significant decrease in remediation effect and soil improvement ability.

[0060] Relying solely on chemical agents (Comparative Example 1) can only temporarily fix heavy metals while damaging the soil ecosystem, failing to achieve the integrated effect of heavy metal detoxification and soil remediation.

[0061] 6. Wide range of applicable scenarios and stronger adaptability to pollution levels: Examples 1-3 respectively covered lightly polluted farmland, moderately polluted irrigated farmland, and heavily polluted copper-zinc industrial clay sites, and all achieved stable compliance under different soil types and pollution loads; while traditional single remediation schemes are only suitable for lightly polluted single sites, and the remediation effect of high-concentration complex heavy metal sites is seriously unsatisfactory.

[0062] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A soil heavy metal passivation bioremediation system mediated by Bacillus belyssus, characterized in that, The system comprises a compound functional microbial agent, an organic synergistic carrier, and a passivation auxiliary agent. The compound functional microbial agent is a highly active solid powder of Bacillus vesicles brevis that has been acclimatized to heavy metal gradients. The organic synergistic carrier is a compound system of well-rotted organic fertilizer, humic acid, and straw charcoal. The passivation auxiliary agent is a compound system of citric acid-modified potassium dihydrogen phosphate, nano-diatomaceous earth, and chelated amino acids. The components of the remediation system, by mass parts, are: 20-35 parts of Bacillus vesicles brevis powder, 50-65 parts of organic synergistic carrier, and 10-20 parts of passivation auxiliary agent.

2. The Bacillus belyss-mediated soil heavy metal passivation bioremediation system according to claim 1, characterized in that, The *Bacillus berleis* strain is a dominant strain that has been acclimatized under a combined stress gradient of cadmium, lead, copper, and zinc. The *Bacillus berleis* powder has an effective viable count of ≥20 billion CFU / g, a contamination rate of ≤0.5%, and a moisture content of ≤8%. The strain tolerates heavy metal concentrations ranging from 0 to 120 mg / kg for cadmium, 0 to 800 mg / kg for lead, 0 to 500 mg / kg for copper, and 0 to 1000 mg / kg for zinc.

3. The Bacillus belyss-mediated soil heavy metal passivation bioremediation system according to claim 1, characterized in that, The preparation method of the Bacillus belyss powder includes: (1) Activation of strain: The low-temperature preserved Bacillus belye strain was inoculated into solid LB medium and cultured at 32℃ inverted for 24h to obtain single colonies. The dominant single colonies were selected for slant subculturing twice to ensure the stability of the strain activity. (2) Heavy metal gradient acclimatization: The activated strains were inoculated into LB liquid medium containing gradient complex heavy metals (cadmium 5-120 mg / kg, lead 50-800 mg / kg, copper 5-500 mg / kg, zinc 50-1000 mg / kg), and acclimatized by shaking at 32℃ and 200 r / min. The concentration of heavy metals was gradually increased, and the acclimatization was carried out for 5 generations. High heavy metal resistant dominant strains were screened. (3) Liquid fermentation culture: The domesticated dominant strain was inoculated into the liquid fermentation medium at an inoculation rate of 5%. The medium formula was: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 8 g / L glucose, and natural pH. The culture conditions were 30-35℃, 180-220 r / min shaking culture for 24-36 h to obtain a fermentation stock solution with a viable count ≥8 billion CFU / mL. (4) Preparation of bacterial powder: The fermentation liquid and sterile diatomaceous earth are mixed evenly at a volume-to-mass ratio of 3:

1. The low-temperature vacuum drying process at -40℃ is adopted, the vacuum degree is controlled at 0.08~0.09MPa, and the drying time is 12~16h. After drying, the material is crushed through an 80-mesh standard sieve, sealed and stored in the dark to obtain highly active Bacillus vesiculosus bacterial powder.

4. The Bacillus belyss-mediated soil heavy metal passivation bioremediation system according to claim 1, characterized in that, The organic enhancement carrier, by mass parts, comprises: 30-40 parts of well-rotted livestock and poultry organic fertilizer, 10-15 parts of humic acid, and 10-15 parts of straw charcoal; the well-rotted livestock and poultry organic fertilizer is a high-temperature aerobic decomposition product with a decomposition degree ≥90% and an organic matter content ≥45%; the humic acid is a mineral-derived high-purity humic acid with a purity ≥95%; and the straw charcoal is a corn straw carbonization product produced at 500-600℃ in an anaerobic high-temperature environment with a pore size of 20-50nm and a specific surface area ≥180m² / g.

5. The Bacillus belyss-mediated soil heavy metal passivation bioremediation system according to claim 1, characterized in that, The passivation auxiliary agent, by mass parts, comprises: 4-8 parts modified potassium dihydrogen phosphate, 3-6 parts nano-diatomite, and 3-6 parts chelated amino acids; the modified potassium dihydrogen phosphate is a citric acid surface-modified product with a water-soluble phosphorus content ≥52%; the nano-diatomite has a particle size of 20-50 nm and a porosity ≥85%; and the chelated amino acids are plant-derived complex amino acid chelates with a total amino acid content ≥30%.

6. The Bacillus belyss-mediated soil heavy metal passivation bioremediation system according to claim 1, characterized in that, The preparation process of the remediation system is as follows: accurately weigh the organic synergist and passivation auxiliary agent according to the ratio, put them into a horizontal mixer, and stir at low speed for 10 minutes to achieve premixing; then add Bacillus vesiculosus powder, stir at low speed for 15 minutes, and operate at room temperature, away from light and in a closed environment throughout the process to avoid inactivation of the strain. After mixing evenly, seal and package, and store at room temperature in a cool place to obtain the finished remediation system.

7. The Bacillus belyss-mediated soil heavy metal passivation bioremediation system according to claim 1, characterized in that, This includes an in-situ repair process, which comprises the following steps: Step S1, Soil pretreatment: Remove stones, weeds, plastic debris and other waste from the soil surface manually or mechanically, and use a deep tiller to plow the soil to a depth of 20-30cm, break up soil clods and level the land. Adjust the soil moisture content to 18%-25% by watering or sun drying to ensure that the soil is loose and breathable, providing good conditions for bacterial colonization and material mixing. Step S2, Application of the remediation system: Adjust the application amount according to the degree of soil pollution. Apply 80-100 kg per acre for lightly polluted soil and 100-120 kg per acre for moderately to heavily polluted soil. Apply manually or by mechanized application with a fertilizer spreader to ensure uniform distribution of materials in the surface soil. Step S3, Soil Mixing and Curing: Deeply till the soil again to a depth of 15-25cm to thoroughly mix the remediation system with the 0-30cm topsoil layer, eliminating material stratification; cover the soil with plastic film for 20-30 days to maintain moisture, strictly controlling the soil moisture content to 20%-28% and the ambient temperature to 15-35℃ during the curing period, avoiding strong sunlight exposure and soil drought, to provide a stable environment for the reproduction and metabolism of Bacillus belyssus; Step S4, Post-maintenance and reuse: After the maintenance period ends, remove the mulch film and allow it to air dry naturally for 3-5 days. Once the soil moisture content has dropped to the normal level in the field, crop planting or site restoration can be carried out.

8. The Bacillus belyss-mediated soil heavy metal passivation bioremediation system according to claim 7, characterized in that, In step S3, the optimal maintenance parameters are: maintenance temperature 20-30℃, soil moisture content 22%-25%, and maintenance time 25-28 days.

9. The application of the Bacillus vesiculosus-mediated soil heavy metal passivation bioremediation system according to any one of claims 1-8 in the in-situ passivation remediation and ecological improvement of single and complex heavy metal contaminated soils of lead, cadmium, copper and zinc in farmland, mining areas and industrial sites.