A method for repairing cadmium pollution in soil by using hyperaccumulating plants and acid-producing bacterial fermentation liquid

Abstract

The application discloses a method for repairing cadmium contaminated soil by using hyperaccumulator and acid-producing bacteria fermentation liquor, and belongs to the technical field of soil remediation. The method comprises the following steps: firstly, screening and identifying the acid-producing bacteria, collecting samples from different cadmium contaminated areas, and determining the excellent strains through treatment, screening and rescreening; secondly, preparing the acid-producing bacteria fermentation liquor, and optimizing the culture process according to the strain characteristics; thirdly, comprehensively analyzing the cadmium contaminated soil, and pretreating the texture and pH; then, accurately mixing the fermentation liquor with the soil according to the soil condition, and adding the synergist; subsequently, selecting suitable plant varieties for planting, and monitoring the plant and soil conditions by doing well the moisture, fertilization and rhizosphere regulation; finally, monitoring and regulating the microbial community by using layered sampling and advanced technology. The application comprehensively utilizes the advantages of each link, effectively solves many deficiencies of the existing microbial remediation technology in the treatment of cadmium contaminated soil, improves the remediation efficiency and success rate, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of soil remediation technology, specifically to a method for remediating cadmium-contaminated soil using a combination of hyperaccumulating plants and acid-producing bacteria fermentation broth. Background Technology

[0002] With rapid industrial development, increased mining, and wastewater irrigation, soil cadmium pollution has become increasingly serious. Cadmium is a highly toxic heavy metal element with high mobility and bioavailability in soil. It easily accumulates in organisms through the food chain, posing a significant threat to ecosystem balance and human health. For example, long-term consumption of crops grown in cadmium-contaminated soil can damage organs such as the kidneys and bones, leading to serious diseases.

[0003] Traditional methods for cadmium remediation in soil include physical remediation, chemical remediation, and phytoremediation. Physical remediation methods, such as topsoil replacement and soil replacement, can reduce cadmium content in soil to some extent, but they suffer from problems such as large-scale engineering, high costs, and the potential for secondary pollution. Chemical remediation methods, such as the use of chemical leaching agents, may damage soil structure and soil ecological balance, affecting the sustainable use of soil. Phytoremediation, while having the advantage of being environmentally friendly, is limited in efficiency due to the speciation of cadmium in the soil, thus restricting its practical application.

[0004] Microbial remediation technology has gradually gained attention as an emerging remediation method. Acid-producing bacteria can generate organic acids through metabolism, altering the physicochemical properties of soil, transforming the form of cadmium in the soil, and increasing the bioavailability of cadmium. This, in turn, promotes the absorption and accumulation of cadmium by plants, enhancing the effectiveness of phytoremediation. However, current methods for using acid-producing bacteria in conjunction with hyperaccumulating plants for soil cadmium remediation still have many shortcomings, such as insufficient precision and efficiency in screening acid-producing bacteria, imperfect fermentation broth preparation processes, lack of targeted pretreatment of contaminated soil, and insufficient systematic monitoring and control of the microbial community during the remediation process. These problems limit the large-scale application of phytoremediation technology in the treatment of soil cadmium pollution. Summary of the Invention

[0005] The purpose of this invention is to provide a method for remediating cadmium-contaminated soil by combining hyperaccumulating plants with acid-producing bacteria fermentation broth, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for remediating cadmium-contaminated soil using a combination of hyperaccumulating plants and acid-producing bacteria fermentation broth, characterized by comprising the following steps:

[0007] S100: Screening and identification of acid-producing bacteria. Through specific sample collection, processing, screening culture medium preparation, screening and re-screening procedures, acid-producing bacteria with excellent soil cadmium activation ability are identified.

[0008] S200: Preparation of acid-producing bacteria fermentation broth, based on the selected strains through activation culture, seed culture, fermentation culture and subsequent treatment, to obtain high-quality and high-efficiency fermentation broth;

[0009] S300: Pretreatment of contaminated soil. After a comprehensive analysis and assessment of cadmium-contaminated soil, operations such as texture improvement, pH adjustment, and microbial activation are carried out according to the soil conditions to make the soil texture uniform.

[0010] S400: The mixture of acid-producing bacteria fermentation broth and soil is prepared by accurately calculating the amount of fermentation broth to be added based on the degree of cadmium pollution and texture of the soil. Special equipment and processes are used for mixing, and synergists are added. The mixture is then placed in a suitable environment.

[0011] S500: Planting and remediation process, selecting suitable plant varieties, pre-treating seeds or seedlings and planting them at a reasonable density, implementing water and fertilizer management and rhizosphere regulation, monitoring plant and soil conditions and adjusting remediation measures;

[0012] S600: Microbial community monitoring and regulation, employing stratified sampling and advanced technologies to monitor microbial communities, analyze their structure and function, and regulate imbalanced communities.

[0013] Preferably, the screening and identification of the S100 acid-producing bacteria specifically includes: collecting soil and sediment samples from areas with different levels of cadmium pollution, such as industrial pollution areas, mine peripheries, and wastewater-irrigated farmland; recording ecological environment information such as soil pH, humidity, temperature, and vegetation type; transporting the samples to the laboratory at low temperature in an insulated box with ice packs; removing impurities by passing the samples through a 2-3 mm sieve in the laboratory; suspending the samples in 0.1 M phosphate buffer (pH 7.0-7.2); shaking at 150-200 rpm for 30-60 minutes; preparing a screening culture medium by adding a cadmium source (such as CdCl2) with a final concentration of 10-20 mg / L, calcium gluconate with a concentration of 0.5%-1%, and a cadmium enrichment solution with a concentration of 0.1%-0.3% to the basal culture medium. The initial pH was adjusted to 7.5-8.0 using citric acid. The treated samples were then diluted using a serial dilution method. A suitable amount of the diluted solution was spread onto screening medium plates, with 3-5 replicates per dilution. The plates were incubated at 28-30℃ for 2-4 days. Colonies whose surrounding medium turned yellow or showed a clear zone were selected. The preliminarily screened colonies were inoculated into liquid medium containing 30-50 mg / L cadmium. The pH was measured using an ion-selective electrode method, and the cadmium ion concentration was measured using atomic absorption spectrometry. Strains that significantly reduced the pH of the culture medium and significantly increased the cadmium ion concentration were screened. The physiological and biochemical characteristics of the re-screened strains were analyzed, including Gram staining, oxidase and catalase tests, and the ability to utilize different carbon, nitrogen, and phosphorus sources.

[0014] Preferably, the preparation of the S200 acid-producing bacteria fermentation broth specifically includes: selecting the high cadmium activation capacity strain screened according to claim 2, preparing an activation culture medium according to its optimal growth conditions, containing 0.5%-2% of the optimal carbon source, 0.1%-0.5% of the optimal nitrogen source, phosphorus source, and 1-5 ppm of trace element solution, inoculating the preserved strain into the activation culture medium, and culturing it at 28-32℃ and 150-200 rpm for 12-24 hours; in the seed culture stage, adding a vitamin mixture (such as vitamin B complex) with a concentration of 0.01%-0.05% to the culture medium containing peptone (1%), beef extract (0.3‰), NaCl (0.5‰), and pH 7.2-7.4, and inoculating the activated strain into the seed culture medium at an inoculation rate of 5%-10%. In the culture medium, shake and culture for 18-24 hours at 30-32℃ and 200-250rpm. During fermentation, add 0.2%-0.5% disodium succinate and 0.05M-0.1M dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer system to the culture medium. Inoculate the seed culture at a rate of 10%-15% and culture for 48-72 hours at 30-32℃ and 200-250rpm. Detect the pH, organic acid content, and cell concentration of the fermentation broth every 12 hours to determine the optimal fermentation endpoint. After the fermentation endpoint is reached, centrifuge at 8000-10000rpm and 4-6℃ for 15-20 minutes to remove cells and macromolecular impurities. Then filter the solution through a 0.22-0.45 filter membrane for sterilization and collect the filtrate for storage in a sterile container at 4-8℃.

[0015] Preferably, the pretreatment of the S300 contaminated soil specifically includes: determining soil texture, organic matter content, total nitrogen, available nitrogen, available phosphorus, and pH value of cadmium-contaminated soil samples; analyzing the cadmium occurrence forms and other heavy metal element contents using X-ray fluorescence spectroscopy (XRF) and inductively coupled plasma mass spectrometry (ICP-MS); if the soil texture is heavy clay, adding 2%-5% biochar and 3%-6% perlite by weight of the soil, mixing evenly, and adjusting the soil moisture to 60%-70% of field capacity, and incubating at 25-30℃ for 1-2 weeks; if the soil pH value is higher than 7.5, adding 0.5-2 grams of sulfur powder per kilogram of soil for acidification treatment; if the soil pH value is lower than 6.0, adding 1-3 grams of slaked lime per kilogram of soil for neutralization treatment; after treatment, the soil needs to be tilled and mixed, and the moisture content needs to be adjusted to a suitable range; 0.1%-0.3% of a microbial activator containing beneficial microorganisms such as Bacillus and Pseudomonas and their metabolites can be added; finally, the soil is air-dried and passed through a 2-3 mm sieve.

[0016] Preferably, the mixing of the S400 acid-producing bacteria fermentation broth with the soil specifically includes: according to the degree of cadmium pollution, texture, and analysis results of the physicochemical properties of the soil after pretreatment, 200-300 ml of fermentation broth is added per kilogram of heavily cadmium-polluted and heavy-textured soil, and 100-150 ml of fermentation broth is added per kilogram of lightly polluted and loose-textured soil; a mixer with multi-directional rotary stirring function and a combination of spiral and arc-shaped stirring blades is used, the soil is first placed in the mixing tank, the fermentation broth is slowly added dropwise, and the mixture is stirred at a low speed of 50-80 rpm for 15-20 minutes, and then stirred at a high speed of 100-150 rpm for 30-40 minutes; 0.05%-0.1% sodium carboxymethyl cellulose (CMC) is added during the mixing process; after mixing, the soil and fermentation broth mixture is transferred to a breathable and waterproof reaction container made of a new type of material, and placed in an environmental room with a temperature of 25-30℃ and a relative humidity of 70%-80%, and turned over once every 12 hours.

[0017] Preferably, the S500 plant planting and remediation process specifically includes: selecting plant varieties that are highly enriched in cadmium and adapted to the local environment based on the previous soil conditions and pollution status; before sowing or transplanting, the seeds are treated with cadmium-containing 10-50 ppm auxin, 5-20 ppm cytokinin, and 10-10 ppm cytokinin. 6 Soak seedlings in a solution of beneficial bacteria such as rhizobia and arbuscular mycorrhizal fungi (CFU / mL) for 4-6 hours. Soak seedling roots in this solution for 30-60 minutes. Plant seedlings at a spacing of 20-30 cm between plants and 30-40 cm between rows, and water thoroughly. Install a soil moisture sensor. When soil moisture content falls below the optimal growth limit for plants (e.g., 60% field capacity), use drip irrigation or micro-sprinkler irrigation, adding a 100-200 times diluted amino acid water-soluble fertilizer to the irrigation water. Before planting, apply 2-3 kg of organic fertilizer per square meter and 50-80 g of nitrogen-phosphorus-potassium fertilizer per square meter. For example, slow-release fertilizer of 15:15:15 is applied as foliar fertilizer and rhizosphere fertilizer during key periods such as seedling stage, jointing stage, and flowering stage. The foliar fertilizer contains 0.1%-0.3% trace elements such as zinc, iron, and manganese and 0.01%-0.05% brassinolide. The rhizosphere fertilizer is a compound fertilizer of potassium dihydrogen phosphate and potassium humate with a concentration of 0.2%-0.5%. 0.5-1mM disodium ethylenediaminetetraacetate and 0.1%-0.3% seaweed extract are added to the rhizosphere soil regularly. The plant growth status and cadmium content changes are closely monitored throughout the growth cycle, and the remediation measures and management strategies are adjusted according to the monitoring results.

[0018] Preferably, the S600 microbial community monitoring and regulation specifically includes: collecting soil samples in the remediation area using a stratified sampling method, stratifying samples at depths of 0-10cm, 10-20cm, and 20-30cm (excluding rhizosphere and non-rhizosphere samples), collecting samples with a sterile sampler, storing them in an ice box, and sending them to the laboratory as soon as possible; monitoring the microbial community using metagenomics and metabolomics methods; constructing an ecological network based on the monitoring data to analyze the community structure and function, and identifying key microbial populations and species; when the number of key microorganisms decreases, using a special encapsulation technique to encapsulate highly active acid-producing bacteria, nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and other beneficial microorganisms in biodegradable polymer microspheres, adding nutrients such as sucrose and amino acids and plant hormone analogs to the microspheres, applying them at a rate of 50-100 grams per square meter and mixing them by shallow tilling to a depth of 5-10cm; compost with an organic matter content of not less than 30% and a specific surface area of ​​not less than 500m² can be added. 2 / g of biochar improves the living environment of microorganisms; intercropping or rotation of plant varieties that can secrete specific signaling substances or nutrients regulates the microbial community in the remediation area.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] 1. Based on the optimal growth conditions of the selected strains, activation culture, seed culture, and fermentation culture were carried out, with the addition of specific inducers and buffering substances. This not only improved the growth activity and metabolic efficiency of the strains but also promoted the production of more organic acids with strong cadmium-chelating ability by acid-producing bacteria. Simultaneously, strict determination of the fermentation endpoint and subsequent treatment steps ensured the high quality and stability of the fermentation broth, enabling it to play a better role in the activation and remediation of cadmium in soil.

[0021] 2. After a comprehensive analysis and assessment of cadmium-contaminated soil, improvements and adjustments were made to address issues such as soil texture and pH value, and microbial activators were added. This improved soil aeration, water retention, and microbial activity, reduced the toxic effects of cadmium on microorganisms and plants, created a favorable soil environment for acid-producing bacteria and plant growth, and improved the overall efficiency of the remediation process.

[0022] 3. The amount of fermentation broth added is precisely calculated based on soil characteristics, and special stirring equipment and mixing processes are used, combined with the use of synergists, to achieve thorough and efficient mixing of the fermentation broth and soil. This ensures that the active ingredients in the fermentation broth can fully act on the cadmium in the soil, maximizing the activation effect of the fermentation broth on cadmium and enhancing the remediation effect.

[0023] 4. Selecting suitable plant varieties and pre-treating them, implementing precise water and fertilizer management and rhizosphere regulation techniques, while closely monitoring plant growth and cadmium content changes. This promoted plant growth and cadmium absorption and accumulation in cadmium-contaminated soil, accelerated the soil cadmium remediation process, and improved the success rate and stability of remediation.

[0024] 5. Stratified sampling and advanced monitoring technologies were employed to comprehensively monitor and analyze the microbial community, enabling timely detection of community imbalances and the implementation of effective control measures. By supplementing microbial inoculants, adding soil conditioners, and utilizing plant-microbe interactions, the stability and activity of the microbial community were maintained, ensuring the efficient operation of the entire remediation system and guaranteeing that the microbial community consistently supports the continuous advancement of cadmium remediation efforts. Attached Figure Description

[0025] Figure 1 This is a schematic flowchart of a method for remediating cadmium-contaminated soil using a combination of hyperaccumulating plants and acid-producing bacteria fermentation broth, according to the present invention. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] This invention presents a technical solution: a method for remediating cadmium-contaminated soil using a combination of hyperaccumulating plants and acid-producing bacteria fermentation broth, mainly comprising:

[0028] S100: Screening and identification of acid-producing bacteria;

[0029] S200: Preparation of acid-producing bacteria fermentation broth;

[0030] S300: Pretreatment of contaminated soil;

[0031] S400: A mixture of acid-producing bacteria fermentation broth and soil;

[0032] S500: Planting and Restoration Process;

[0033] S600: Microbial community monitoring and regulation;

[0034] In one preferred embodiment of the present invention, in the screening and identification of acid-producing bacteria in S100, firstly, soil and sediment samples from areas with varying degrees of cadmium pollution are collected, covering industrial pollution areas, mine perimeters, and wastewater-irrigated farmland. Simultaneously, the ecological environment of the samples is recorded in detail, including soil pH, humidity, temperature, and vegetation type, as these factors affect the community structure and function of microorganisms.

[0035] After collection, samples are quickly placed in an insulated box with ice packs for transport to ensure they reach the laboratory as soon as possible under low temperature conditions. In the laboratory, the samples undergo preliminary processing: larger stones and impurities are removed by passing them through a 2-3 mm sieve. Then, they are suspended in a sterile buffer solution (such as 0.1 M phosphate buffer, pH 7.0-7.2) and shaken on a shaker at 150-200 rpm for 30-60 minutes to ensure thorough dispersion of microorganisms.

[0036] When preparing the screening medium, an appropriate amount of cadmium source (such as CdCl2, with a final concentration of 10-20 mg / L) is added to the basal medium to simulate a polluted environment. Simultaneously, substances that induce acid production, such as calcium gluconate (0.5%-1%) and fulvic acid (0.1%-0.3%), are added. Calcium gluconate acts as a carbon source and inducer to promote the production of organic acids by acid-producing bacteria, while fulvic acid mimics the organic components in natural soil, stimulating the metabolic activity of acid-producing bacteria. Furthermore, the initial pH of the medium is adjusted to 7.5-8.0 to create a suitable acid-producing environment for the bacteria.

[0037] The treated samples were diluted to different folds using a serial dilution method. A suitable amount of the diluted solution was then spread onto screening medium plates, with 3-5 replicates for each dilution. The incubation conditions were 28-30℃, and the incubation time depended on colony growth, generally 2-4 days. During incubation, colony morphology was observed, and colonies showing clear signs of acid production, such as yellowing of the surrounding medium or the appearance of a clear zone, were selected.

[0038] The initially selected colonies underwent further screening. They were inoculated into liquid culture medium containing a higher concentration of cadmium (e.g., 30-50 mg / L) and cultured for a period of time. Changes in pH and cadmium ion concentration were measured. pH was determined using an ion-selective electrode method, and cadmium ion concentration was determined using atomic absorption spectrometry. Strains that significantly reduced the pH of the culture medium and significantly increased the cadmium ion concentration were selected, indicating that they possessed strong acid-producing and cadmium-activating abilities.

[0039] Physiological and biochemical characteristics of the strains after secondary screening were analyzed. In addition to routine tests such as Gram staining, oxidase, and catalase, the focus was on detecting their ability to utilize different carbon, nitrogen, and phosphorus sources. Culture media with multiple carbon sources (such as glucose, sucrose, and lactose), nitrogen sources (such as ammonium nitrate, ammonium chloride, and urea), and phosphorus sources (such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate) were set up to observe the growth of the strains and determine their optimal nutrient conditions, providing a basis for subsequent fermentation culture.

[0040] Through the above comprehensive screening and identification process, acid-producing bacteria with excellent soil cadmium activation capabilities can be efficiently screened out, providing a strong guarantee of microbial resources for subsequent remediation work.

[0041] In the preferred embodiment of the present invention, in the preparation of the acid-producing bacteria fermentation broth in S200, after the screening and identification of acid-producing bacteria are completed, acid-producing bacteria strains with high cadmium activation ability are selected for fermentation broth preparation.

[0042] First, the selected strains were activated. A specialized activation medium was prepared based on the optimal growth conditions observed during the screening process. This medium contained appropriate proportions of carbon source (e.g., the optimal carbon source determined in S100, with its concentration precisely controlled between 0.5% and 2%), nitrogen source (e.g., the optimal nitrogen source concentration maintained at 0.1% to 0.5%), and phosphorus source (to ensure the supply of phosphorus required for the strain's growth and metabolism). A small amount of trace element solution (e.g., containing trace elements such as iron, manganese, and zinc, at a concentration of 1-5 ppm) was also added to promote rapid growth and metabolic activity of the strains. The preserved strains were inoculated into the activation medium and cultured at a suitable temperature (e.g., 28-32℃) and shaking speed (150-200 rpm) for 12-24 hours to restore the strains to a good growth state.

[0043] During the seed culture stage, the traditional seed culture medium was optimized. In addition to containing peptone (1%), beef extract (0.3‰), NaCl (0.5‰), and a suitable pH (7.2-7.4), a certain amount of vitamin mixture (such as B vitamins, concentration 0.01%-0.05%) was added. The addition of vitamins helps improve the strain's stress resistance and metabolic efficiency, enhancing its stability during subsequent fermentation. Activated chrysanthemum plants were inoculated into the seed culture medium at an inoculation rate of 5%-10%, and cultured in a shaker at 30-32°C with shaking at 200-250 rpm for 18-24 hours to ensure sufficient cell concentration and good activity in the seed culture.

[0044] During fermentation, the composition of the fermentation broth was finely adjusted. Based on the acid-producing characteristics and cadmium activation mechanism exhibited by the acid-producing bacteria in S100, specific inducers and buffers were added. 0.2%-0.5% disodium succinate was added as an inducer to further stimulate the acid-producing bacteria to produce more organic acids with strong cadmium-chelating ability. Simultaneously, an appropriate amount of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer system (concentration 0.05M-0.1M) was added to maintain pH stability during fermentation and prevent a sharp drop in pH due to excessive acid production, which would inhibit bacterial growth. The seed culture was inoculated into the fermentation broth at an inoculum volume of 10%-15% and cultured at 30-32°C and 200-250 rpm for 48-72 hours. During this process, samples were taken every 12 hours to test the pH value, organic acid content (analyzed by high performance liquid chromatography), and cell concentration (determined by hemocytometer or spectrophotometer) of the fermentation broth, and growth curves and metabolite change curves were plotted to determine the optimal fermentation endpoint.

[0045] Once fermentation reaches its predetermined endpoint, the fermentation broth is immediately processed. First, it is centrifuged at 4-6°C for 1520 minutes using a high-speed centrifuge (8000-10000 rpm) to remove bacterial cells and large molecular impurities. Then, the supernatant is filtered through a 0.22-0.45 mm filter membrane to ensure the fermentation broth is free of live bacteria, preventing the introduction of foreign microorganisms in subsequent soil remediation applications. The filtered fermentation broth is collected, placed in a sterile container, and stored at 4-8°C for later use to maintain its activity and stability, preparing it for subsequent mixing with contaminated soil for cadmium activation remediation. Through these innovative steps and optimized measures, a high-quality, high-efficiency acid-producing bacterial fermentation broth is prepared for the remediation of cadmium-contaminated soil.

[0046] In one preferred embodiment of the present invention, in step S300: the pretreatment of contaminated soil, before pretreatment, a comprehensive analysis and evaluation of the collected cadmium-contaminated soil samples is performed. In addition to determining conventional basic physicochemical properties such as soil texture (using the international soil texture classification standard, determining the ratio of sand, silt, and clay particles through mechanical analysis), organic matter content (accurately determined using the potassium dichromate oxidation-external heating method), total nitrogen (using the Kjeldahl method), available nitrogen (alkaline diffusion method), available phosphorus (molybdenum-antimony colorimetric method), and pH value (glass electrode method), advanced X-ray fluorescence spectroscopy (XRF) and inductively coupled plasma mass spectrometry (ICP-MS) techniques are used to conduct a detailed analysis of the occurrence forms of cadmium in the soil and the content of other heavy metal elements that may affect the remediation effect.

[0047] Based on the initial condition of the soil, targeted pretreatment should be carried out. If the soil texture is relatively heavy and has poor aeration, it can be improved by adding appropriate amounts of biochar (2%-5% of soil mass) and perlite (3%-6% of soil mass). Biochar has a rich porous structure and a large specific surface area, which can improve soil aeration and water retention. At the same time, its surface functional groups can also adsorb some heavy metal ions, reducing the toxic effects of cadmium on microorganisms and plants. Perlite helps to increase soil porosity, improving soil aeration and water permeability. After thoroughly mixing biochar and perlite with the soil, adjust the soil moisture to 60%-70% of field capacity and cultivate it in a greenhouse or indoor temperature-controlled environment (temperature set at 25-30℃) for 1-2 weeks to allow the soil microbial community to gradually adapt to the new environment and promote the initial stabilization of the soil ecosystem.

[0048] For soils with excessively high or low pH values, appropriate adjustments should be made. When the soil pH is above 7.5, sulfur powder should be used for acidification. The amount of sulfur powder to add should be calculated based on the soil's buffering capacity and initial pH value, generally 0.5-2 grams per kilogram of soil. After evenly spreading the sulfur powder on the soil surface, thoroughly till and mix it. Then, maintain soil moisture within a certain range and regularly monitor changes in soil pH until it drops to a suitable range (6.5-7.5). If the soil pH is below 6.0, add an appropriate amount of slaked lime for neutralization. The amount of slaked lime added depends on the soil acidity, typically 1-3 grams per kilogram of soil. Again, after tilling, mixing, and moisture control, stabilize the soil pH within a suitable range.

[0049] In soil pretreatment, microbial activators can be added to enhance the activity and diversity of soil microorganisms. Microbial preparations containing various beneficial microorganisms (such as Bacillus and Pseudomonas) and their metabolites (such as amino acids, vitamins, and polysaccharides) are added to the soil at a ratio of 0.1%-0.3%. These microorganisms can participate in soil material cycling and energy conversion, helping to improve the soil ecological environment and enhance soil cleanliness. Furthermore, they synergistically work with subsequently added acid-producing bacteria fermentation broth to promote cadmium activation and remediation.

[0050] After the above series of pretreatment steps, the soil is air-dried and passed through a 2-3 mm sieve to remove residual impurities and larger particles, making the soil texture uniform and providing a good foundation for subsequent mixing with acid-producing bacteria fermentation liquid and planting, thus ensuring the smooth progress of the soil remediation process.

[0051] In one preferred embodiment of the present invention, in step S400, the mixing of the acid-producing bacteria fermentation broth with the soil is carried out after the pretreatment of the contaminated soil. First, the amount of acid-producing bacteria fermentation broth to be added is precisely calculated based on the degree of cadmium contamination, soil texture, and the physicochemical properties analyzed after pretreatment. For heavily contaminated soils with a relatively heavy texture, the proportion of fermentation broth is appropriately increased, generally 200-300 ml per kilogram of soil; while for lightly contaminated soils with a loose texture, 100-150 ml per kilogram of soil is added to ensure that the active ingredients in the fermentation broth can fully act on the cadmium in the soil.

[0052] Special mixing equipment and processes are employed during the mixing process. A mixer with multi-directional rotary mixing function is used, whose mixing blades are designed with a unique combination of spiral and arc shapes, generating a complex flow field during mixing to achieve efficient and uniform mixing of the fermentation broth and soil particles. First, the soil is placed in the mixing tank of the mixer, then the acid-producing bacteria fermentation broth is slowly added dropwise while the mixer is started and stirred at a low speed of 50-80 rpm for 15-20 minutes to allow the fermentation broth to initially contact and disperse with the soil. Next, the stirring speed is increased to 100-150 rpm, and stirring continues for 30-40 minutes to ensure that the fermentation broth fully penetrates the pores of the soil particles, undergoing a full chemical reaction and physical adsorption with cadmium ions.

[0053] To further enhance the interaction between the fermentation broth and the soil, a certain amount of synergist is added during the mixing process. Adding 0.05%-0.1% sodium carboxymethyl cellulose (CMC) can increase the adhesion and stability of the fermentation broth in the soil, preventing it from flowing away or settling too quickly and prolonging its action time. Simultaneously, CMC can regulate the soil's aggregate structure, improve soil aeration and permeability, and facilitate microbial activity and cadmium migration and transformation in the soil.

[0054] After mixing, the soil and fermentation broth mixture is transferred to a specially designed reaction vessel. This vessel is made of a novel, breathable yet waterproof material, ensuring an adequate oxygen supply for soil microorganisms while preventing excessive moisture loss. The reaction vessel is placed in a temperature- and humidity-controlled indoor environment, with the temperature maintained at 25-30°C and the relative humidity at 70%-80%, providing suitable conditions for the growth and metabolism of acid-producing bacteria and promoting their continuous activation of cadmium. Furthermore, the mixture is gently agitated every 12 hours to ensure a more even distribution of substances within the soil, guaranteeing a sustained and effective reaction between the fermentation broth and the cadmium in the soil.

[0055] Through the above innovative mixing steps and measures, the acid-producing bacteria fermentation broth is fully and efficiently mixed with the contaminated soil, maximizing the activation effect of the fermentation broth on cadmium in the soil and laying a good foundation for the subsequent remediation process.

[0056] In the preferred embodiment of the present invention, in S500: the plant planting and restoration process, after the acid-producing bacteria fermentation liquid is mixed with the soil and properly placed in a suitable environment, the plant planting and restoration stage begins.

[0057] First, based on a comprehensive assessment of soil conditions and pollution status, plant varieties with high cadmium accumulation capacity and adapted to the local environment are selected, such as certain hyperaccumulating plants or local plants with good remediation potential after screening. Before sowing or transplanting, plant seeds or seedlings are pretreated. Seeds are soaked in a solution containing plant growth hormones (such as appropriate amounts of auxin and cytokinin, at concentrations of 10-50 ppm and 5-20 ppm respectively) and microbial agents (containing beneficial bacteria that promote root growth and enhance plant resistance, such as rhizobia and arbuscular mycorrhizal fungi, at a concentration of 10-10 FU / mL) for 4-6 hours to promote seed germination and root development, enhancing the plant's survival ability in cadmium-contaminated soil. For seedlings, their roots are soaked in the same microbial agent solution for 30-60 minutes before transplanting to ensure the roots carry sufficient beneficial microorganisms. When planting, arrange the plants according to a reasonable planting density. Based on the growth characteristics of the plants and the soil fertility, generally maintain a plant spacing of 20-30 cm and a row spacing of 30-40 cm to ensure that each plant receives sufficient nutrients, water, and light, while avoiding excessive competition. After planting, immediately water thoroughly to ensure that the plant roots are in close contact with the soil, which is beneficial for the roots to absorb water and nutrients.

[0058] Implement precise water management strategies during plant growth. Install soil moisture sensors to monitor soil moisture content in real time and irrigate according to plant water requirements and soil moisture conditions. When soil moisture content falls below the lower limit for optimal plant growth (e.g., 60% field capacity), supplemental irrigation using drip irrigation or micro-sprinkler irrigation techniques should be employed to avoid flooding that could damage soil structure and cause cadmium migration and diffusion. Simultaneously, add appropriate amounts of organic nutrient solution (e.g., amino acid water-soluble fertilizer diluted 100-200 times) to the irrigation water to provide additional nutritional support for plants, promoting plant growth and cadmium absorption.

[0059] Regular fertilization management is essential. In addition to applying appropriate amounts of base fertilizer, such as organic fertilizer and slow-release fertilizer, before planting (2-3 kg of organic fertilizer per square meter, and 50-80 g of slow-release fertilizer per square meter based on a nitrogen-phosphorus-potassium ratio of 15:15:15), foliar and rhizosphere fertilizers should be applied during key growth stages, such as the seedling stage, jointing stage, and flowering stage. Foliar fertilizers can be solutions containing trace elements (such as zinc, iron, and manganese, at a concentration of 0.1%-0.3%) and plant growth regulators (such as brassinolide, at a concentration of 0.01%-0.05%), sprayed onto the plant leaves to enhance photosynthesis and stress resistance. Rhizosphere fertilizers should be water-soluble fertilizers rich in phosphorus, potassium, and organic matter (such as potassium dihydrogen phosphate and potassium humate compound fertilizer, at a concentration of 0.2%-0.5%), applied in a ring around the plant roots to promote root growth and cadmium absorption and translocation.

[0060] To enhance plants' ability to absorb and tolerate cadmium, rhizosphere regulation techniques can be employed. Regularly add appropriate amounts of chelating agents (such as disodium EDTA, at a concentration of 0.5-1 mM) and biostimulants (such as seaweed extract, at a concentration of 0.1%-0.3%) to the rhizosphere soil. The chelating agent can form complexes with cadmium in the soil that are more easily absorbed by plants, increasing the bioavailability of cadmium; the biostimulant can activate the plant's physiological metabolic processes, enhancing the plant's tolerance and absorption capacity for cadmium.

[0061] Throughout the entire plant growth cycle, plant growth is closely monitored, including morphological indicators such as plant height, leaf color and quantity, and stem diameter, as well as physiological indicators such as photosynthetic efficiency and stomatal conductance, measured using equipment such as chlorophyll fluorometers and photosynthesis meters. Simultaneously, plant and rhizosphere soil samples are collected regularly, and atomic absorption spectrometry and inductively coupled plasma mass spectrometry are used to analyze cadmium content in the aboveground and underground parts of the plants, as well as changes in the form and content of cadmium in the soil. Based on the monitoring results, remediation measures and management strategies are adjusted promptly to ensure the efficient implementation of the phytoremediation process until the expected soil cadmium remediation target is achieved.

[0062] In the preferred embodiment of the present invention, in S600: microbial community monitoring and regulation, the stability and activity of the microbial community play a key role in the restoration effect during the plant growth restoration process, so it is necessary to continuously monitor and effectively regulate it.

[0063] First, soil samples were collected in the remediation area where plants had already been planted using a stratified sampling method. In addition to the conventional rhizosphere and non-rhizosphere soil sampling, soil samples were also collected according to depth (e.g., 0-10cm, 10-20cm, 20-30cm, etc.) to more accurately understand the changes in soil microbial communities at different depths. An appropriate amount of soil was collected from each sampling point using a sterile sampler, mixed thoroughly, and immediately placed in an ice box for preservation before being transported to the laboratory for analysis as soon as possible.

[0064] A variety of advanced technologies are employed to comprehensively monitor the microbial community. Metagenomics technology, through whole-genome sequencing analysis of soil microorganisms, not only provides species information but also offers in-depth insights into the composition and expression of their functional genes, particularly those related to cadmium metabolism, organic acid production, and plant growth promotion. Simultaneously, metabolomics methods are combined to detect changes in the types and amounts of microbial metabolites in the soil, such as organic acids, amino acids, and vitamins. These metabolites have a significant impact on cadmium transformation and plant growth.

[0065] Detailed community structure and function analysis was conducted based on monitoring data. Bioinformatics tools were used to construct the ecological network of the microbial community, analyzing the relationships between different microbial populations, such as symbiosis, competition, and synergistic effects. Core microbial populations playing a crucial role in cadmium pollution remediation, as well as key species significantly influencing community stability, were identified.

[0066] When an imbalance in the microbial community or a weakening of the function of beneficial microorganisms is detected, timely regulatory measures should be implemented. If the number of key microorganisms such as acid-producing bacteria decreases, highly efficient microbial agents can be prepared specifically to supplement them. These agents utilize a special encapsulation technology to encapsulate highly active acid-producing bacteria and other beneficial microorganisms (such as ammonia-fixing bacteria and phosphate-solubilizing bacteria) in biodegradable polymer microspheres. Appropriate amounts of nutrients (such as sucrose amino acids) and growth promoters (such as plant hormone analogs) are added to the microspheres to ensure the slow release and continuous growth of microorganisms in the soil. Apply the agent evenly to the soil surface at a dosage of 50-100 grams per square meter, and combine with shallow tillage (5-10 cm depth) to ensure thorough mixing with the soil.

[0067] To improve the living environment for microorganisms, soil conditioners can be added. Applying compost rich in organic matter, with an organic matter content of no less than 30%, can increase soil fertility and water retention, providing microorganisms with abundant carbon and energy sources. Simultaneously, adding an appropriate amount of biochar, with a specific surface area of ​​no less than 500 m² / g, can adsorb heavy metal ions due to its porous structure, reducing cadmium toxicity to microorganisms. It can also provide attachment sites for microorganisms, promoting their colonization and growth in the soil.

[0068] Furthermore, regulation can be achieved using the principles of plant-microbe interactions. Selecting plant varieties capable of secreting specific signaling substances or nutrients and intercropping or rotating them in the remediation area can stimulate the growth and activity of beneficial microorganisms, promote the recovery and stability of the microbial community, enhance the overall remediation system's ability to remediate cadmium-contaminated soil, and ensure that the microbial community remains in a state conducive to cadmium remediation.

[0069] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for remediating cadmium-contaminated soil using a combination of hyperaccumulating plants and acid-producing bacteria fermentation broth, characterized in that, Includes the following steps: S100: Screening and identification of acid-producing bacteria. Through specific sample collection, processing, screening culture medium preparation, screening and re-screening procedures, acid-producing bacteria with excellent soil cadmium activation ability are identified. S200: Preparation of acid-producing bacteria fermentation broth, based on the selected strains through activation culture, seed culture, fermentation culture and subsequent treatment, to obtain high-quality and high-efficiency fermentation broth; S300: Pretreatment of contaminated soil. After a comprehensive analysis and assessment of cadmium-contaminated soil, operations such as texture improvement, pH adjustment, and microbial activation are carried out according to the soil conditions to make the soil texture uniform. S400: The mixture of acid-producing bacteria fermentation broth and soil is prepared by accurately calculating the amount of fermentation broth to be added based on the degree of cadmium pollution and texture of the soil. Special equipment and processes are used for mixing, and synergists are added. The mixture is then placed in a suitable environment. S500: Planting and remediation process, selecting suitable plant varieties, pre-treating seeds or seedlings and planting them at a reasonable density, implementing water and fertilizer management and rhizosphere regulation, monitoring plant and soil conditions and adjusting remediation measures; S600: Microbial community monitoring and regulation, employing stratified sampling and advanced technologies to monitor microbial communities, analyze their structure and function, and regulate imbalanced communities.

2. The repair method according to claim 1, characterized in that, The screening and identification of S100 acid-producing bacteria specifically includes: collecting soil and sediment samples from areas with different levels of cadmium pollution, such as industrial pollution zones, mine peripheries, and wastewater-irrigated farmland; recording ecological environment information such as soil pH, humidity, temperature, and vegetation type; transporting the samples to the laboratory at low temperature in an insulated box with ice packs; removing impurities by passing the samples through a 2-3 mm sieve in the laboratory; suspending the samples in 0.1 M phosphate buffer (pH 7.0-7.2) and shaking at 150-200 rpm for 30-60 minutes; preparing a screening culture medium by adding a cadmium source (such as CdCl2) at a final concentration of 10-20 mg / L, calcium gluconate at a concentration of 0.5%-1%, and fulvic acid at a concentration of 0.1%-0.3%. The initial pH was adjusted to 7.5-8.

0. The treated samples were diluted using a serial dilution method, and an appropriate amount of the diluted solution was spread onto screening medium plates. Each dilution was replicated 3-5 times. The plates were incubated at 28-30℃ for 2-4 days, and colonies whose surrounding medium turned yellow or showed a clear zone were selected. The preliminarily screened colonies were inoculated into liquid medium containing 30-50 mg / L cadmium. The pH was measured using an ion-selective electrode method, and the cadmium ion concentration was measured using atomic absorption spectrometry. Strains that significantly reduced the pH of the culture medium and significantly increased the cadmium ion concentration were screened. The physiological and biochemical characteristics of the re-screened strains were analyzed, including Gram staining, oxidase, catalase tests, and the ability to utilize different carbon, nitrogen, and phosphorus sources.

3. The repair method according to claim 1, characterized in that, The preparation of the S200 acid-producing bacteria fermentation broth specifically includes: selecting the high cadmium activation capacity strain screened according to claim 2, preparing an activation culture medium according to its optimal growth conditions, containing 0.5%-2% of the optimal carbon source, 0.1%-0.5% of the optimal nitrogen source, phosphorus source, and 1-5 ppm of trace element solution; inoculating the preserved strain into the activation culture medium and culturing it at 28-32℃ and 150-200 rpm for 12-24 hours; in the seed culture stage, adding a vitamin mixture (such as B vitamins) with a concentration of 0.01%-0.05% to the culture medium containing peptone (1%), beef extract (0.3‰), NaCl (0.5‰), and pH 7.2-7.4; and inoculating the activated strain into the seed culture medium at an inoculation rate of 5%-10%. The culture was shaken at 30-32℃ and 200-250 rpm for 18-24 hours. During fermentation, 0.2%-0.5% disodium succinate and 0.05M-0.1M dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer system were added to the culture medium. The seed culture was inoculated at a rate of 10%-15%. The culture was then incubated at 30-32℃ and 200-250 rpm for 48-72 hours. The pH, organic acid content, and cell concentration of the fermentation broth were measured every 12 hours to determine the optimal fermentation endpoint. After the fermentation endpoint was reached, the culture was first centrifuged at 8000-10000 rpm and 4-6℃ for 15-20 minutes to remove cells and macromolecular impurities. Then, the culture was filtered through a 0.22-0.45 filter membrane for sterilization. The filtrate was collected and stored in a sterile container at 4-8℃.

4. The repair method according to claim 1, characterized in that, The pretreatment of S300 contaminated soil specifically includes: determining soil texture, organic matter content, total nitrogen, available nitrogen, available phosphorus, and pH value of cadmium-contaminated soil samples; analyzing the cadmium occurrence forms and other heavy metal content using X-ray fluorescence spectroscopy (XRF) and inductively coupled plasma mass spectrometry (ICP-MS); if the soil texture is heavy clay, adding 2%-5% biochar and 3%-6% perlite by weight of the soil, mixing thoroughly, and adjusting the soil moisture to 60%-70% of field capacity, and incubating at 25-30℃ for 1-2 weeks; if the soil pH value is higher than 7.5, adding 0.5-2 grams of sulfur powder per kilogram of soil for acidification treatment; if the soil pH value is lower than 6.0, adding 1-3 grams of slaked lime per kilogram of soil for neutralization treatment; after treatment, the soil must be tilled and mixed, and the moisture content adjusted to a suitable range; 0.1%-0.3% of a microbial activator containing beneficial microorganisms such as Bacillus and Pseudomonas and their metabolites can be added; finally, the soil is air-dried and passed through a 2-3 mm sieve.

5. The repair method according to claim 1, characterized in that, The mixing of the S400 acid-producing bacteria fermentation broth with the soil specifically includes: based on the degree of cadmium pollution, texture, and pre-treatment physicochemical properties of the soil, 200-300 ml of fermentation broth is added per kilogram for heavily cadmium-polluted and heavy clay soil, and 100-150 ml of fermentation broth is added per kilogram for lightly polluted and loose soil; using a mixer with multi-directional rotary stirring function and a combination of spiral and arc-shaped stirring blades, the soil is first placed in the mixing tank, and the fermentation broth is slowly added dropwise, stirred at a low speed of 50-80 rpm for 15-20 minutes, and then stirred at a high speed of 100-150 rpm for 30-40 minutes; 0.05%-0.1% sodium carboxymethyl cellulose (CMC) is added during the mixing process; after mixing, the soil and fermentation broth mixture is transferred to a breathable but waterproof reaction container made of a new type of material, placed in an environmental room with a temperature of 25-30℃ and a relative humidity of 70%-80%, and turned over every 12 hours.

6. The repair method according to claim 1, characterized in that, The S500 plant planting and remediation process specifically includes: selecting plant varieties that are highly enriched in cadmium and adapted to the local environment based on the previous soil conditions and pollution status; before sowing or transplanting, the seeds are treated with a solution containing 10-50 ppm auxin, 5-20 ppm cytokinin, and 10-10 ppm cadmium. 6 Soak seedlings in a solution of beneficial bacteria such as rhizobia and arbuscular mycorrhizal fungi (CFU / mL) for 4-6 hours. Soak seedling roots in this solution for 30-60 minutes. Plant seedlings at a spacing of 20-30 cm between plants and 30-40 cm between rows, and water thoroughly. Install a soil moisture sensor. When soil moisture content falls below the optimal growth limit for plants (e.g., 60% field capacity), use drip irrigation or micro-sprinkler irrigation, adding a 100-200 times diluted amino acid water-soluble fertilizer to the irrigation water. Before planting, apply 2-3 kg of organic fertilizer per square meter and 50-80 g of nitrogen-phosphorus-potassium fertilizer per square meter. For example, slow-release fertilizer of 15:15:15 is applied as foliar fertilizer and rhizosphere fertilizer during key periods such as seedling stage, jointing stage, and flowering stage. The foliar fertilizer contains 0.1%-0.3% trace elements such as zinc, iron, and manganese and 0.01%-0.05% brassinolide. The rhizosphere fertilizer is a compound fertilizer of potassium dihydrogen phosphate and potassium humate with a concentration of 0.2%-0.5%. 0.5-1mM disodium ethylenediaminetetraacetate and 0.1%-0.3% seaweed extract are added to the rhizosphere soil regularly. The plant growth status and cadmium content changes are closely monitored throughout the growth cycle, and the remediation measures and management strategies are adjusted according to the monitoring results.

7. The repair method according to claim 1, characterized in that, The S600 microbial community monitoring and regulation specifically includes: collecting soil samples in the remediation area using a stratified sampling method. In addition to rhizosphere and non-rhizosphere sampling, samples are collected at depths of 0-10cm, 10-20cm, and 20-30cm. Samples are collected using a sterile sampler, stored in an ice box, and sent to the laboratory as soon as possible. Metagenomics and metabolomics methods are used to monitor the microbial community. Based on the monitoring data, an ecological network is constructed to analyze the community structure and function, identifying key microbial populations and species. When the number of key microorganisms decreases, highly active acid-producing bacteria, nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and other beneficial microorganisms are encapsulated in biodegradable polymer microspheres using a special encapsulation technique. Nutrients such as sucrose, amino acids, and plant hormone analogs are added to the microspheres. The microspheres are applied at a rate of 50-100 grams per square meter and shallowly tilled to a depth of 5-10cm for mixing. Compost with an organic matter content of not less than 30% and a specific surface area of ​​not less than 500m² can be added. 2 / g of biochar improves the living environment of microorganisms; intercropping or rotation of plant varieties that can secrete specific signaling substances or nutrients regulates the microbial community in the remediation area.

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