A soil microbial agent for repairing hexavalent chromium and a preparation method and application thereof

Abstract

The application belongs to the technical field of microbial application, and provides a hexavalent chromium remediation microbial agent for soil, a preparation method and application thereof, wherein the microbial agent takes Bacillus paramycoides C6 with a preservation number of CGMCC No. 20390 as a functional strain, and takes modified calcium-based bentonite as a immobilization carrier. The microbial agent has a good remediation effect in soil, can significantly reduce the content of hexavalent chromium in soil, and has a long persistence and strong environmental adaptability.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, specifically to a soil hexavalent chromium remediation microbial agent, its preparation method, and its application. Background Technology

[0002] Chromium pollution has seriously threatened agricultural production, human health, and ecological balance. The accumulation of chromium slag and the discharge of chromium wastewater lead to the accumulation of chromium in surrounding soil and water bodies, which exists stably as hexavalent chromium (Cr(VI)) and trivalent chromium (Cr(III)) compounds. This alters the composition of polluted water and soil, disrupts microbial community structure, and severely reduces soil fertility. Cr(VI) can enter plant cells from the soil, inhibiting seed germination, seedling growth, and subsequent development; it can also enter animals and humans through the water and food chain, causing neurological dysfunction and inducing cancer. Cr(VI) compounds have been classified as Group 1 carcinogens by the World Health Organization. As a major global industrial manufacturing base, my country relies heavily on chromium salts and their related products as key chemical raw materials. The situation of chromium pollution in water and soil is severe, and chromium has been listed as one of the key heavy metal pollutants threatening national safety. Therefore, developing efficient and environmentally friendly hexavalent chromium remediation technologies has become an urgent need in the field of environmental governance.

[0003] Cr(III) is poorly soluble in water, an essential micronutrient for life, non-carcinogenic, and its toxicity is only one-thousandth that of Cr(VI). Therefore, reducing Cr(VI) to Cr(III) is a more thorough and effective remediation pathway than adsorption and enrichment. In recent years, interdisciplinary technologies for Cr(VI) pollution control, combining physics, chemistry, materials science, and biology, have emerged, including photocatalytically assisted two-dimensional Co-Al layered double hydroxide / TiO2 heterostructure materials, modified Fe-C microelectrolysis processes, adsorption using organobentonite nanocomposites, in-situ fixation of ferrous ions, and chromium recovery using oxalic acid. However, these methods generally suffer from high costs, high energy and chemical reagent consumption, stringent requirements for environmental conditions such as pH, temperature, and light, complex operation, and the need for real-time monitoring, limiting their practical application.

[0004] Phytoremediation can be used to treat chromium-contaminated sites. The synergistic effect of plants and bacteria can increase the accumulation of Cr(VI) in plants, but chromium-accumulating plants still require secondary treatment and are difficult to achieve efficient in-situ detoxification. Microorganisms participate in the natural cycle of matter and maintain the balance of ecosystems, making them important functional organisms for environmental remediation. Since the discovery of bacteria with Cr(VI) reduction capabilities in the 1970s, microbial remediation has been recognized as the most promising Cr(VI) removal technology due to its advantages such as being green, efficient, low-cost, capable of in-situ treatment, and highly sustainable.

[0005] In chromium-contaminated environments, bacteria can evolve various anti-chromium stress mechanisms, mainly including reduced uptake, increased efflux, damage repair, extracellular reduction, adsorption and fixation, and intracellular reduction. Cr(VI) can enter the bacterial cell via sulfate ion channels due to their structural similarity to sulfate. Bacteria can reduce uptake through channel deformation, pump Cr(VI) out of the cell via ChrA efflux proteins, or cope with oxidative stress through SOD enzymes and DNA damage repair. However, these are all passive anti-chromium mechanisms and cannot effectively remove Cr(VI) from the environment. Bacterial cell wall functional groups such as carboxyl, amino, hydroxyl, phosphate, and sulfhydryl groups can non-enzymatically reduce Cr(VI), but the extracellular reduction efficiency is low, only effective when the Cr(VI) concentration is below 10 mg / L. Bacteria can also adsorb and accumulate Cr(VI) on the cell wall or intracellularly, but this is difficult to recover and cannot achieve pollutant detoxification, still posing a risk of secondary pollution.

[0006] Intracellular chromium reductases such as YieF, NfsA, ChrR, NemA, and ChrT have been found in strains of Bacillus, Escherichia coli, Pseudomonas, and Serratia, providing a core functional basis for microbial remediation. However, existing chromium-reducing strains generally suffer from poor tolerance to hexavalent chromium and low reduction efficiency, which seriously restricts the large-scale application of microbial remediation technology. Summary of the Invention

[0007] In view of this, embodiments of the present invention provide a soil hexavalent chromium remediation microbial agent, its preparation method and application. The present invention uses the patented strain C6 as the functional bacteria and adopts modified inorganic carrier calcium-based bentonite combined with cross-linking fixation technology to prepare a solid microbial agent that can efficiently remediate soil hexavalent chromium, significantly improving the stability, colonization ability and remediation efficiency of the agent, and can achieve rapid, long-term and low-cost remediation of heavily hexavalent chromium contaminated soil.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A soil remediation microbial agent using hexavalent chromium, wherein the agent uses Bacillus paramycoides C6 (CGMCC No. 20390) as the functional strain (deposited at the China General Microbiological Culture Collection Center on July 17, 2020, and classified as Bacillus sp.), and uses modified calcium-based bentonite as the immobilization carrier; the modified calcium-based bentonite is subjected to high-temperature treatment, metal cation modification, and chitosan coating modification in sequence.

[0010] Furthermore, the metal cation modification includes at least one of Fe(III), Al(III), Co(II) or Na(I) modification.

[0011] The present invention also provides a method for preparing the above-mentioned soil hexavalent chromium remediation microbial agent, comprising the following steps:

[0012] Functional strain culture: Bacillus paramycoides C6 was optimized and cultured in a medium containing hexavalent chromium. Bacterial cells in the logarithmic growth phase were collected and a bacterial suspension was prepared.

[0013] Modification of calcium-based bentonite: After sieving, washing, and drying, calcium-based bentonite is placed in a muffle furnace and slowly heated to 450-550℃ for 1-2 hours. After slow cooling, it is removed, crushed, and sieved to obtain pretreated calcium-based bentonite. The pretreated calcium-based bentonite is added to a metal cation solution and stirred at a constant temperature of 25-35℃ for 3.5-4.5 hours. After settling, the supernatant is discarded, and the bentonite is washed with deionized water until neutral. After drying, metal cation-modified calcium-based bentonite is obtained. The metal cation-modified calcium-based bentonite is dispersed in a chitosan solution and ultrasonically treated for 25-35 minutes. It is then concentrated to a paste, freeze-dried, crushed, and sieved to obtain modified calcium-based bentonite.

[0014] Immobilization: The obtained modified calcium-based bentonite was mixed with the bacterial suspension and adsorbed by shaking; calcium chloride solution was added for cross-linking treatment;

[0015] Molding and drying: The cross-linked system is mixed with sterile water and freeze-dried to obtain bacterial powder.

[0016] Furthermore, the concentration of hexavalent chromium in the culture medium is 200 mg / L, and the optimized culture time is 12-18 h.

[0017] Furthermore, the bacterial suspension is prepared by washing the bacterial cells with phosphate buffer, centrifuging, resuspending, and adjusting the bacterial concentration to 1×10⁻⁶. 10 CFU / mL.

[0018] Furthermore, the solid-liquid ratio of the pretreated calcium-based bentonite to the metal cation solution is 1g:10mL, and the concentration of the metal cation solution is 0.5mol / L.

[0019] Furthermore, in the calcium-based bentonite modification step, the drying temperature is 105°C during metal cation modification.

[0020] Furthermore, the solid-liquid ratio of the metal cation modified calcium-based bentonite to the chitosan solution is 1 g: 5 mL.

[0021] Furthermore, the solid-liquid ratio of the modified calcium-based bentonite to the bacterial suspension is 1:2 (g / mL).

[0022] Furthermore, the oscillation adsorption temperature was 23~27℃, and the time was 0.8~1.2h.

[0023] Furthermore, the amount of calcium chloride solution added is 30% of the mass of the modified calcium-based bentonite carrier.

[0024] Furthermore, this invention also provides the application of the above-mentioned microbial agents in the remediation of hexavalent chromium contaminated soil.

[0025] The beneficial effects of the embodiments of the present invention compared with the prior art include:

[0026] 1. The strain exhibits extremely high tolerance and provides specific and efficient repair.

[0027] This invention utilizes Bacillus paramycoides C6 (accession number CGMCC No. 20390), which exhibits a minimum inhibitory concentration (MIC) of up to 2500 mg / L against hexavalent chromium (Cr), and can grow and function normally in heavily contaminated soil. After induction culture with hexavalent chromium, the strain converts highly toxic Cr(VI) into non-toxic Cr(III), eliminating the need for secondary treatment and achieving complete remediation.

[0028] 2. Scientific carrier modification significantly improves loading and colonization capabilities.

[0029] A three-step modification of calcium-based bentonite using high-temperature activation, metal cation modification, and chitosan coating significantly improves the specific surface area, pore volume, and pore size of the carrier, providing numerous attachment sites for the bacteria. The chitosan-Ca²⁺ cross-linking network enhances the stability of the bacteria and improves the soil microenvironment, enabling functional strains to rapidly colonize and survive in the soil for a long time, making them less susceptible to elimination by indigenous microorganisms.

[0030] 3. The microbial agent is stable at room temperature and is convenient to store and apply.

[0031] The powder was prepared using a freeze-drying process and stored at room temperature in a cool place for one year, maintaining a viable bacterial count and spore count of 2 × 10⁻⁶. 9 With a CFU / g or higher, the spore production rate is consistently above 95%. The microbial agent is easy to store and transport, and can be directly applied by spreading and tilling, requiring no complicated equipment. It is suitable for in-situ / ex-situ remediation.

[0032] 4. High remediation efficiency, rapid attainment of standards even in cases of severe pollution.

[0033] At the optimal application rate of 50 g / kg soil, the removal rate of hexavalent chromium contaminated soil reached 99.99% in 30 days; for heavily contaminated soil, the removal rate was ≥97% in 90 days, and the screening value for Class II land use in GB 36600-2018 could be reached in 180 days, with stable and reliable remediation effect.

[0034] 5. Long-lasting protection against secondary pollution; effective for a long time after a single application.

[0035] Functional strains can colonize the soil for a long time, continuously express functional genes, and can quickly initiate remediation in the face of secondary hexavalent chromium pollution. A single application can achieve long-term stable treatment, significantly reducing the cost of repeated investment.

[0036] 6. Green and environmentally friendly, low cost, and widely applicable.

[0037] The microbial agent uses natural mineral calcium-based bentonite as a carrier, leaving no chemical residues, damaging soil structure, or causing secondary pollution. It also improves soil pH and enhances the soil's microecological environment. With low raw material costs, its cost-effectiveness is significantly superior to physical and chemical remediation technologies, making it widely applicable to the remediation of various hexavalent chromium-contaminated soils, including industrial sites, chromium slag dumps, and farmland. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 This describes the change in the expression level of the bcr005 gene over time under different initial Cr(VI) concentrations according to the present invention.

[0040] Figure 2 This invention describes the change in the expression level of the bcb765 gene over time under different initial Cr(VI) concentrations.

[0041] Figure 3 The removal rate of hexavalent chromium Cr(VI) by strain C6 under different initial Cr(VI) concentrations according to the present invention;

[0042] Figure 4 These are the growth curves of strain C6 under different initial Cr(VI) concentrations according to the present invention;

[0043] Figure 5 This is a scanning electron microscope image of strain C6 of the present invention;

[0044] Figure 6 These are scanning electron microscope (SEM) images of the calcium-based bentonite before and after modification according to this invention. Detailed Implementation

[0045] The present invention will be described more clearly below with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the function of the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0046] To make the objectives, technical solutions, and advantages of the present invention clearer, specific embodiments will be described below in conjunction with the accompanying drawings.

[0047] Example 1

[0048] This invention provides a soil hexavalent chromium remediation microbial agent, and the preparation method of the soil hexavalent chromium remediation microbial agent includes the following steps:

[0049] Functional strain culture: Bacillus paramycoides C6 was cultured in a medium containing 200 mg / L hexavalent chromium for 15 h. Cells in the logarithmic growth phase were collected, washed twice with phosphate buffer (pH 7.0), centrifuged at 5000 rpm for 10 min, resuspended, and the bacterial concentration adjusted to 1×10⁻⁶. 10 CFU / mL was used to prepare a bacterial suspension.

[0050] Modification of calcium-based bentonite: After being sieved through a 200-mesh sieve, washed three times with anhydrous ethanol, and dried at 80℃, calcium-based bentonite was placed in a muffle furnace and slowly heated to 500℃ at a rate of 10℃ / min, and kept at this high temperature for 1.5 hours. After slow cooling, it was removed, pulverized, and sieved through a 200-mesh sieve to obtain pretreated calcium-based bentonite. The pretreated calcium-based bentonite was added to a 0.5 mol / L FeCl3 solution at a solid-liquid ratio of 1:10 (g / mL), stirred at 30℃ for 4 hours, allowed to settle and precipitate, the supernatant was discarded, and the bentonite was washed with deionized water until neutral. It was then dried at 105℃ to obtain Fe. 3+ Modified calcium-based bentonite, with Fe 3+ Modified calcium-based bentonite was dispersed in a 2% chitosan solution (containing 1% acetic acid) at a solid-liquid ratio of 1:5 (g / mL), ultrasonically treated (power 300W) for 30 min, rotary evaporated at 60℃ to a paste, freeze-dried, and then pulverized through a 100-mesh sieve to obtain modified calcium-based bentonite.

[0051] Immobilization: The modified calcium-based bentonite was mixed with the bacterial suspension at a ratio of 1:2 (g / mL), and the mixture was shaken at 150 rpm for 1 h at 25 ℃ for adsorption. At this point, the bacterial cells were completely adsorbed onto the carrier. Then, 30% (w / v) of the modified calcium-based bentonite carrier mass of a 1% CaCl2 solution was added, stirred until homogeneous, and allowed to stand for 30 min for cross-linking treatment to form chitosan-Ca... 2+ Cross-linked networks enhance the adhesion stability of bacterial strains;

[0052] Molding and Drying: The cross-linked immobilized carrier was mixed with sterile water at a ratio of 3:1 (g / g), and freeze-dried for 6 hours, controlling the moisture content at 8%~10%, to obtain the bacterial powder. At this point, the viable count was ≥5×10⁻⁶. 9CFU / g, spore count ≥4.5×10 9 The number of cells / g is 90%, and the spore production rate reaches 90%.

[0053] Example 2

[0054] This invention provides a soil hexavalent chromium remediation microbial agent, and the preparation method of the soil hexavalent chromium remediation microbial agent includes the following steps:

[0055] Functional strain culture: Bacillus paramycoides C6 was cultured in a medium containing 200 mg / L hexavalent chromium for 12 h. Cells in the logarithmic growth phase were collected, washed twice with phosphate buffer (pH 7.0), centrifuged at 5000 rpm for 10 min, resuspended, and the bacterial concentration adjusted to 1×10⁻⁶. 10 CFU / mL was used to prepare a bacterial suspension.

[0056] Modification of calcium-based bentonite: Calcium-based bentonite was sieved through a 200-mesh sieve, washed three times with anhydrous ethanol, and dried at 80℃. It was then placed in a muffle furnace and slowly heated to 550℃ at a rate of 10℃ / min, held at this high temperature for 1.0 h, slowly cooled, removed, pulverized, and sieved through a 200-mesh sieve to obtain pretreated calcium-based bentonite. The pretreated calcium-based bentonite was added to a 0.5 mol / L FeCl3 solution at a solid-liquid ratio of 1:10 (g / mL), stirred at 30℃ for 4 h, allowed to settle and precipitate, the supernatant was discarded, and the bentonite was washed with deionized water until neutral. It was then dried at 105℃ to obtain Fe. 3+ Modified calcium-based bentonite, with Fe 3+ Modified calcium-based bentonite was dispersed in a 2% chitosan solution (containing 1% acetic acid) at a solid-liquid ratio of 1:5 (g / mL), ultrasonically treated (power 300W) for 30 min, rotary evaporated at 60℃ to a paste, freeze-dried, and then pulverized through a 100-mesh sieve to obtain modified calcium-based bentonite.

[0057] Immobilization: The modified calcium-based bentonite was mixed with the bacterial suspension at a ratio of 1:2 (g / mL), and the mixture was shaken at 150 rpm for 0.8 h at 27 ℃, at which point the bacterial cells were completely adsorbed onto the carrier. Then, 30% (w / v) of the modified calcium-based bentonite carrier mass of a 1% CaCl2 solution was added, stirred until homogeneous, and allowed to stand for 30 min for cross-linking treatment to form chitosan-Ca... 2+ Cross-linked networks enhance the adhesion stability of bacterial strains;

[0058] Molding and drying: The cross-linked and immobilized carrier is mixed with sterile water at a ratio of 3:1 (g / g), freeze-dried for 6 hours, and the moisture content is controlled at 8%~10% to obtain the bacterial agent powder.

[0059] Example 3

[0060] This invention provides a soil hexavalent chromium remediation microbial agent, and the preparation method of the soil hexavalent chromium remediation microbial agent includes the following steps:

[0061] Functional strain culture: Bacillus paramycoides C6 was cultured for 18 h in a medium containing 200 mg / L hexavalent chromium. Cells in the logarithmic growth phase were collected, washed twice with phosphate buffer (pH 7.0), centrifuged at 5000 rpm for 10 min, resuspended, and the bacterial concentration adjusted to 1 × 10⁻⁶. 10 CFU / mL was used to prepare a bacterial suspension.

[0062] Modification of calcium-based bentonite: After being sieved through a 200-mesh sieve, washed three times with anhydrous ethanol, and dried at 80℃, calcium-based bentonite was placed in a muffle furnace and slowly heated to 500℃ at a rate of 10℃ / min, and kept at this high temperature for 1.5 hours. After slow cooling, it was removed, pulverized, and sieved through a 200-mesh sieve to obtain pretreated calcium-based bentonite. The pretreated calcium-based bentonite was added to a 0.5 mol / L FeCl3 solution at a solid-liquid ratio of 1:10 (g / mL), stirred at 25℃ for 4.5 hours, allowed to settle and precipitate, the supernatant was discarded, and the bentonite was washed with deionized water until neutral. It was then dried at 105℃ to obtain Fe. 3+ Modified calcium-based bentonite, with Fe 3+ Modified calcium-based bentonite was dispersed in a 2% chitosan solution (containing 1% acetic acid) at a solid-liquid ratio of 1:5 (g / mL), ultrasonically treated (power 300W) for 30 min, rotary evaporated at 60℃ to a paste, freeze-dried, and then pulverized through a 100-mesh sieve to obtain modified calcium-based bentonite.

[0063] Immobilization: The modified calcium-based bentonite was mixed with the bacterial suspension at a ratio of 1:2 (g / mL), and the mixture was shaken at 150 rpm for 1 h at 25 ℃ for adsorption. At this point, the bacterial cells were completely adsorbed onto the carrier. Then, 30% (w / v) of the modified calcium-based bentonite carrier mass of a 1% CaCl2 solution was added, stirred until homogeneous, and allowed to stand for 30 min for cross-linking treatment to form chitosan-Ca... 2+ Cross-linked networks enhance the adhesion stability of bacterial strains;

[0064] Molding and drying: The cross-linked and immobilized carrier is mixed with sterile water at a ratio of 3:1 (g / g), freeze-dried for 6 hours, and the moisture content is controlled at 8%~10% to obtain the bacterial agent powder.

[0065] Example 4

[0066] This invention provides a soil hexavalent chromium remediation microbial agent, and the preparation method of the soil hexavalent chromium remediation microbial agent includes the following steps:

[0067] Functional strain culture: Bacillus paramycoides C6 was cultured in a medium containing 200 mg / L hexavalent chromium for 15 h. Cells in the logarithmic growth phase were collected, washed twice with phosphate buffer (pH 7.0), centrifuged at 5000 rpm for 10 min, resuspended, and the bacterial concentration adjusted to 1×10⁻⁶. 10 CFU / mL was used to prepare a bacterial suspension.

[0068] Modification of calcium-based bentonite: Calcium-based bentonite was sieved through a 200-mesh sieve, washed three times with anhydrous ethanol, and dried at 80℃. It was then placed in a muffle furnace and slowly heated to 450℃ at a rate of 10℃ / min, held at this high temperature for 2 hours, slowly cooled, removed, pulverized, and sieved through a 200-mesh sieve to obtain pretreated calcium-based bentonite. The pretreated calcium-based bentonite was added to a 0.5 mol / L FeCl3 solution at a solid-liquid ratio of 1:10 (g / mL), stirred at 30℃ for 4 hours, allowed to settle and precipitate, the supernatant was discarded, and the bentonite was washed with deionized water until neutral. It was then dried at 105℃ to obtain Fe. 3+ Modified calcium-based bentonite, with Fe 3+ Modified calcium-based bentonite was dispersed in a 2% chitosan solution (containing 1% acetic acid) at a solid-liquid ratio of 1:5 (g / mL), ultrasonically treated (power 300W) for 30 min, rotary evaporated at 60℃ to a paste, freeze-dried, and then pulverized through a 100-mesh sieve to obtain modified calcium-based bentonite.

[0069] Immobilization: The modified calcium-based bentonite was mixed with the bacterial suspension at a ratio of 1:2 (g / mL), and the mixture was shaken at 150 rpm for 1.2 h at 23 ℃, at which point the bacteria were completely adsorbed onto the carrier. Then, 30% (w / v) of the modified calcium-based bentonite carrier mass of a 1% CaCl2 solution was added, stirred until homogeneous, and allowed to stand for 30 min for cross-linking treatment to form chitosan-Ca... 2+ Cross-linked networks enhance the adhesion stability of bacterial strains;

[0070] Molding and drying: The cross-linked and immobilized carrier is mixed with sterile water at a ratio of 3:1 (g / g), freeze-dried for 6 hours, and the moisture content is controlled at 8%~10% to obtain the bacterial agent powder.

[0071] Example 5

[0072] This invention provides a soil hexavalent chromium remediation microbial agent, and the preparation method of the soil hexavalent chromium remediation microbial agent includes the following steps:

[0073] Functional strain culture: Bacillus paramycoides C6 was cultured in a medium containing 200 mg / L hexavalent chromium for 15 h. Cells in the logarithmic growth phase were collected, washed twice with phosphate buffer (pH 7.0), centrifuged at 5000 rpm for 10 min, resuspended, and the bacterial concentration adjusted to 1×10⁻⁶. 10 CFU / mL was used to prepare a bacterial suspension.

[0074] Modification of calcium-based bentonite: Calcium-based bentonite was sieved through a 200-mesh sieve, washed three times with anhydrous ethanol, and dried at 80℃. It was then placed in a muffle furnace and slowly heated to 500℃ at a rate of 10℃ / min, held at this high temperature for 1.5 hours, slowly cooled, removed, pulverized, and sieved through a 200-mesh sieve to obtain pretreated calcium-based bentonite. The pretreated calcium-based bentonite was added to a 0.5 mol / L FeCl3 solution at a solid-liquid ratio of 1:10 (g / mL), stirred at 35℃ for 3.5 hours, allowed to settle, and the supernatant was discarded. The bentonite was washed with deionized water until neutral and dried at 105℃ to obtain Fe. 3+ Modified calcium-based bentonite, with Fe 3+ Modified calcium-based bentonite was dispersed in a 2% chitosan solution (containing 1% acetic acid) at a solid-liquid ratio of 1:5 (g / mL), ultrasonically treated (power 300W) for 30 min, rotary evaporated at 60℃ to a paste, freeze-dried, and then pulverized through a 100-mesh sieve to obtain modified calcium-based bentonite.

[0075] Immobilization: The modified calcium-based bentonite was mixed with the bacterial suspension at a ratio of 1:2 (g / mL), and the mixture was shaken at 150 rpm for 1 h at 25 ℃ for adsorption. At this point, the bacterial cells were completely adsorbed onto the carrier. Then, 30% (w / v) of the modified calcium-based bentonite carrier mass of a 1% CaCl2 solution was added, stirred until homogeneous, and allowed to stand for 30 min for cross-linking treatment to form chitosan-Ca... 2+ Cross-linked networks enhance the adhesion stability of bacterial strains;

[0076] Molding and drying: The cross-linked and immobilized carrier is mixed with sterile water at a ratio of 3:1 (g / g), freeze-dried for 6 hours, and the moisture content is controlled at 8%~10% to obtain the bacterial agent powder.

[0077] This invention investigated the cell conditions of Bacillus paramycoides C6. The minimum inhibitory concentration (MIC) of Cr(VI) for C6 was 2500 mg / L. After 18 h of optimized liquid culture, C6 showed a 99.9% removal rate of Cr(VI) at 200 mg / L and a 67.3% removal rate at 2000 mg / L, significantly superior to other reported strains. Differential transcriptome sequencing, quantitative real-time PCR, and protein function verification confirmed that the novel chromium reductase BCR005 and the newly identified chromium-binding protein BCB765 are encoded by bcr005 and bcb765 in the genome. At low Cr(VI) concentrations (200 mg / L), the chromium reductase plays a major role; at 500 mg / L, the chromium-binding protein plays a major role; and above 1000 mg / L, both act synergistically to resist chromium stress (e.g., chromium chromium). Figure 1 , 2 ).

[0078] The results of quantitative real-time PCR showed that when Bacillus C6 was induced with an initial concentration of 200 mg / L chromium for 15 h, it produced a large amount of intracellular chromium reductase, while no significant synthesis of chromium-binding protein was observed. Figure 1 , 2 At this point, the strain can reduce 99% of the hexavalent chromium in the culture medium to trivalent chromium, and the bacteria are in the logarithmic growth phase. Figure 3 , 4 ), with no change in appearance ( Figure 5 It is suitable for use as a hexavalent chromium bactericide in environmental remediation and does not require recycling.

[0079] This invention, through induction culture with different initial concentrations of hexavalent chromium and scanning electron microscopy analysis, found that C6 cells showed the best growth at an initial concentration of 200 mg / L hexavalent chromium. At 15 h, the strain was in the logarithmic growth phase, and at this time, only a large amount of chromium reductase was produced in the cells, thus removing hexavalent chromium by reduction.

[0080] This invention also included a screening and comparative study of the carrier selection. After comparing various carriers such as activated carbon, biochar, carboxymethyl cellulose, and polyvinyl alcohol, calcium-based bentonite was ultimately selected as the carrier for preparing the microbial agent. Bentonite is a natural mineral with a high specific surface area, which provides numerous attachment sites for microorganisms, thereby increasing their loading capacity. Its porous structure (mainly micropores and mesopores) effectively accommodates microorganisms and provides sufficient space and a protective environment, which is conducive to their survival and reproduction. Due to its natural adsorption properties, calcium-based bentonite can interact with the surface of microorganisms through electrostatic interactions or van der Waals forces, enhancing the adhesion between microorganisms and the carrier, thus improving the stability and long-lasting effect of the microbial agent. Furthermore, calcium-based bentonite exhibits good dispersibility, stability, mass transfer performance, and mechanical strength in soil, and is not easily decomposed or migrated. Therefore, when used as a carrier, it can effectively maintain a stable loading of microorganisms and continuously release them into the soil environment. Bentonite's low cost, chemical inertness, and low toxicity make it highly suitable for environmentally friendly remediation applications, particularly in areas such as heavy metal pollution control and agricultural microbial fertilizers, where it can safely provide microbial carriers without negatively impacting ecosystems.

[0081] The selected calcium-based bentonite was analyzed by scanning electron microscopy and specific surface area and porosity analysis. The results are as follows: Figure 6 As shown, its specific surface area is 39.7 m². 2 / g, pore volume 0.08cm 3 / g, with a pore size of 4 nm. To further improve its specific surface area and pore size for stable loading of more bacterial cells, this invention modifies it by optimizing its physical structure through high-temperature treatment to enhance adhesion stability; Fe is added. 3+ It provides chemical anchoring sites and nutritional support for microorganisms; then, through chitosan coating, it enhances the biocompatibility of the microenvironment and provides a carbon source for the long-term survival of microorganisms.

[0082] After modification, the modified calcium-based bentonite obtained according to Example 1 of this invention was subjected to BET analysis again, and the specific surface area of ​​the modified calcium-based bentonite was 58.2 m². 2 / g, pore volume 0.14cm 3 / g, with a pore size of 6 nm, combined with scanning electron microscopy analysis results ( Figure 6 It was found that its surface is rougher, which is more conducive to the attachment of bacterial strains; its specific surface area is larger, which is conducive to the attachment of more bacterial cells and spores; and its pore volume and pore size are larger, resulting in higher gas and material transport efficiency.

[0083] Experimental Example 1

[0084] To verify the characteristics of the microbial agent provided by the present invention, the agent prepared according to Example 1 was used for agent stability testing.

[0085] The bacterial agent was packaged in sample bags, 50g / bag, and stored at 4℃, 22℃ and in a cool indoor place for 7, 14, 30, 90, 180 and 360 days respectively. The number of viable bacteria and spores were recorded in Table 1 below.

[0086] Table 1 Stability test of microbial agents

[0087]

[0088] The appearance of the inoculum stored under various conditions showed no significant changes; the inoculum stored in a cool, shaded indoor location occasionally showed moisture when the temperature was higher. After 360 days of storage, both the number of spores and the number of viable cells were greater than 2 × 10⁻⁶. 9 The bacterial count is CFU / g, and the spore count is greater than 95%. The number of viable bacteria and spores is highest when stored at 4℃. Considering factors such as cost, this bacterial agent can be stored in a cool, dark place indoors in a sealed container.

[0089] Experimental Example 2

[0090] To further verify the characteristics of the microbial agent provided by the present invention, a simulation test of the repair effect was conducted using the agent prepared according to Example 1.

[0091] The remediation effect of the microbial agent on hexavalent chromium contaminated soil was simulated and tested in a laboratory square dish. The indoor temperature range was 17–28 °C, and the humidity range was 15%–30%. The soil source is recorded in Table 2, and the various soil indicators are recorded in Table 3. The optimal dosage of the microbial agent was determined using soil sample No. 3 (Table 4).

[0092] Table 2 Soil sources used in each simulation experiment

[0093]

[0094] Table 3 Soil Indicators Used in Each Simulation Experiment

[0095]

[0096] Table 4. Experimental results of optimal application rate of microbial agent

[0097]

[0098] Based on the results in Table 4, the optimal application rate of the bacterial agent (80 g / kg) resulted in the highest number of functional gene copies of the bacterial strain in the soil within 30 days, while the highest removal rate of hexavalent chromium was achieved at an application rate of 100 g / kg. However, considering the production cost of the bacterial agent, and assuming a removal rate of 99.9% of hexavalent chromium from the soil within 30 days, an application rate of 50 g / kg would be sufficient to achieve the soil remediation goal. Furthermore, at this point, the number of functional gene copies of the bacterial strain in the soil would be 81% of that at 14 days, which is 3% lower than when using 80 g / kg. Conversely, at an application rate of 80 g / kg, the number of functional gene copies of the bacterial strain at 30 days is only 63% of that at 14 days, suggesting a potential significant reduction in strain survival rate due to resource scarcity. Therefore, the application rate of this bacterial agent is set at 50 g / kg.

[0099] Soil samples 1-6 were divided into four portions, each weighing 1 kg, and placed in a 10 cm × 15 cm square dish with a thickness of approximately 5 cm. 50 g of fresh inoculant was added to three of each soil sample from samples 1-6, evenly sprinkled on the soil surface, and thoroughly mixed. One soil sample was used as a blank control without inoculant. Four ventilation holes were left. All soil samples were taken for testing at 7, 14, 30, 90, and 180 days. The samples were thoroughly mixed before each sampling, with 5 g taken at a time. Soil pH, hexavalent chromium content, and abundance of the functional gene bcr005 were measured and recorded in Table 5.

[0100] Table 5 Simulation experiment on the remediation effect of microbial agents on hexavalent chromium in contaminated soil

[0101]

[0102] After six months of treatment with the bacterial agent, under different sources, initial hexavalent chromium contamination levels, pH values, and microbial distributions, the abundance of the C6 functional gene bcr005 in strain C6 was consistently higher than 1×10⁻⁶ at 30 days. 6 The number of copies / g was higher than 1×10⁻⁶ at 180 days. 5 The copy number / g indicates that C6 was successfully colonized. The pH of all soils was adjusted to a slightly alkaline level of 7.8-8.5, and the hexavalent chromium content in various severely contaminated soils was remediated to below 5.7 mg / kg, meeting the screening values ​​for Class II land use in GB36600-2018 "Soil Environmental Quality Standard for Construction Land Soil Pollution Risk Control (Trial)". Soil #4 is the source soil of strain C6; therefore, the unique bcr005 functional gene of C6 could be detected in soil #4 without the addition of inoculant, and the colonization of C6 in soil #4 was relatively good.

[0103] The colonization of the target strain is detected by the relative abundance of the functional gene bcr005 because this functional gene is unique to this strain, which can accurately identify and quantify the target strain, and at the same time reflect the functional activity of the inoculant in the soil.

[0104] To further evaluate the long-term effectiveness of the microbial agent under laboratory conditions, hexavalent chromium was re-added to soil #3 at a final concentration of 200 mg / kg 90 days after the agent was applied. The removal of hexavalent chromium was then tested again at 7, 14, 30 and 90 days after the second addition of pollutants (Table 6).

[0105]

[0106] The results of repeated pollution remediation simulation experiments show that after the first batch of pollutants are removed from the soil, strain C6 can successfully colonize and quickly restore its function when the pollutants reappear. Therefore, after the application of the bacterial agent of this invention, it can colonize the soil for a long time and continuously play the role of removing hexavalent chromium.

[0107] This invention proposes a Bacillus C6 immobilization technology based on a modified bentonite mineral inorganic carrier, and utilizes this technology to prepare a hexavalent chromium removal agent for soil remediation. This agent is then applied to the remediation of hexavalent chromium-contaminated soil. The modified bentonite carrier effectively enhances the stability and activity of the microorganisms by immobilizing the bacterial strain. Experimental results show that this agent has excellent remediation effects in soil, significantly reducing the hexavalent chromium content, and exhibits a long-lasting effect and strong environmental adaptability.

[0108] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A soil remediation microbial agent using hexavalent chromium, characterized in that, The bacterial agent uses Bacillus paramycoides C6, with preservation number CGMCCNo.20390, as the functional strain and modified calcium-based bentonite as the immobilization carrier; the modified calcium-based bentonite is subjected to high-temperature treatment, metal cation modification, and chitosan coating modification in sequence.

2. The soil remediation microbial agent for hexavalent chromium according to claim 1, characterized in that, The metal cation modification includes at least one of Fe(III), Al(III), Co(II) or Na(I) modification.

3. A method for preparing a soil remediation microbial agent for hexavalent chromium as described in claim 1 or 2, characterized in that, Includes the following steps: Functional strain culture: Bacillus paramycoides C6 was optimized and cultured in a medium containing hexavalent chromium. Bacterial cells in the logarithmic growth phase were collected and a bacterial suspension was prepared. Modification of calcium-based bentonite: After sieving, washing, and drying, calcium-based bentonite is placed in a muffle furnace and slowly heated to 450-550℃ for 1-2 hours. After slow cooling, it is removed, crushed, and sieved to obtain pretreated calcium-based bentonite. The pretreated calcium-based bentonite is added to a metal cation solution and stirred at a constant temperature of 25-35℃ for 3.5-4.5 hours. After settling, the supernatant is discarded, and the bentonite is washed with deionized water until neutral. After drying, metal cation-modified calcium-based bentonite is obtained. The metal cation-modified calcium-based bentonite is dispersed in a chitosan solution and ultrasonically treated for 25-35 minutes. It is then concentrated to a paste, freeze-dried, crushed, and sieved to obtain modified calcium-based bentonite. Immobilization: The obtained modified calcium-based bentonite was mixed with the bacterial suspension and adsorbed by shaking; calcium chloride solution was added for cross-linking treatment; Molding and drying: The cross-linked system is mixed with sterile water and freeze-dried to obtain bacterial powder.

4. The preparation method according to claim 3, characterized in that, The concentration of hexavalent chromium in the culture medium was 200 mg / L, and the optimized culture time was 12-18 h.

5. The preparation method according to claim 3, characterized in that, The bacterial suspension was prepared by washing the bacterial cells with phosphate buffer, centrifuging, resuspending, and adjusting the bacterial concentration to 1×10⁻⁶. 10 CFU / mL.

6. The preparation method according to claim 3, characterized in that, The solid-liquid ratio of the pretreated calcium-based bentonite to the metal cation solution is 1g:10mL, and the concentration of the metal cation solution is 0.5mol / L.

7. The preparation method according to claim 3, characterized in that, In the calcium-based bentonite modification step, the drying temperature during metal cation modification is 105℃.

8. The preparation method according to claim 3, characterized in that, The solid-liquid ratio of the metal cation modified calcium-based bentonite to the chitosan solution is 1 g: 5 mL.

9. The preparation method according to claim 3, characterized in that, The amount of calcium chloride solution added is 30% of the mass of the modified calcium-based bentonite carrier.

10. The application of the microbial agent according to claim 1 or 2 in the remediation of hexavalent chromium contaminated soil.

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