Heavy metal resistant bacillus velezensis and microbial remediation agent and application

Abstract

The application discloses a heavy metal resistant bacillus velezensis and a microbial repair agent and application thereof. The bacillus velezensis (Bacillus velezensis DBS01) is screened from heavy metal contaminated soil in Dabaoshan mine area of Shaoguan city in Guangdong province, and is preserved in China Center for Type Culture Collection with a preservation number of CCTCC NO: M 20252228. The bacillus velezensis not only has multiple heavy metal resistance capabilities, but also can metabolically secrete organic acids, cellulase, growth hormones and other metabolites in different growth stages, so as to realize multiple functions such as heavy metal solidification, phosphorus dissolution, potassium dissolution and plant growth promotion. The bacillus velezensis DBS01 is inoculated into a culture medium containing straws, calcium superphosphate is added, and agar and sodium alginate are added, and the mixture containing the bacillus velezensis DBS01 is freeze-dried, so that the mine heavy metal contaminated soil can be repaired in a high-efficiency, low-cost and environment-friendly manner, and the ecological function recovery of the soil can be effectively promoted.

Description

Technical Field

[0001] This invention relates to heavy metal resistant Bacillus belye, and particularly to a heavy metal resistant Bacillus belye, its microbial remediation agent, and its application. Background Technology

[0002] Mining is a vital economic activity for resource acquisition, but it is also a major anthropogenic factor causing damage to soil ecosystems. Large-scale mining activities, especially open-pit mining, strip vegetation and topsoil, leading to soil erosion, compaction, reduced porosity, and the introduction of toxic heavy metals such as lead (Pb), arsenic (As), mercury (Hg), copper (Cu), zinc (Zn), and cadmium (Cd). These heavy metals enter the environment through dust deposition and acidic drainage, directly poisoning soil organisms and inhibiting enzyme activity, and threatening human health through the biomagnification effect of the food chain. Furthermore, acidic mine drainage generated from the oxidation of associated sulfides (such as pyrite) further exacerbates soil acidification, increasing the solubility and bioavailability of heavy metals, leading to nutrient leaching and loss of ecological functions.

[0003] Currently, remediation methods for heavy metal contaminated soil in mining areas mainly include physical, chemical, and biological methods. While physical and chemical methods are relatively fast, they have limitations such as high cost and the potential for secondary pollution. In contrast, bioremediation, especially microbial remediation technology, is considered a green and sustainable solution due to its low cost, environmental friendliness, and ability to be implemented in situ. Among these, Bacillus species have become a hot topic in microbial remediation technology for heavy metal contaminated soil in mining areas because of their ability to form highly resilient spores and their strong survival ability in harsh environments. Bacillus velezensis, a beneficial bacterium that has received widespread attention in recent years, plays a significant role in promoting plant growth and controlling plant diseases. However, its application potential in the remediation of heavy metal contaminated soil, especially for complex heavy metal pollution, has not been fully explored and proven. Existing technologies have the following significant limitations and gaps:

[0004] (1) Narrow resistance spectrum and low tolerance threshold: Chinese invention CN120758429A reported a heavy metal-tolerant Bacillus belye, but it is only resistant to Cd, Pb, Cr and Ni, and the growth of the strain is significantly inhibited when the Cd concentration exceeds 4.0 mg / Kg. This indicates that the strain is difficult to cope with the high concentration and multi-element complex pollution environment commonly found in mining areas.

[0005] (2) The strains have limited functions and lack comprehensive remediation capabilities: Chinese invention CN119913085A discloses a microbial compound preparation that can decompose cellulose, but the Bacillus belysin used in it does not have the ability to decompose cellulose; Chinese invention CN119709511A reports a strain isolated from plants that is tolerant to various environmental pollutants, but it has not been confirmed that it has specific heavy metal resistance or remediation functions. These strains are all unable to achieve the synergistic remediation goals of immobilizing heavy metals, activating soil nutrients, and promoting plant growth.

[0006] (3) The preparation process of the microbial agent damages the activity of microorganisms and results in poor environmental adaptability: Chinese invention CN 119639613A uses a drying process of 42℃~45℃ when preparing the microbial agent, which significantly reduces the activity of microorganisms, making it difficult for the microbial agent to exert its function after being applied to the soil; while Chinese invention CN119632050A uses the traditional sodium alginate embedding method, the main purpose of which is to use the antibacterial effect of the strain to prevent and control plant diseases, without considering the challenge of how to maintain high activity and high stability in mining soils under heavy metal stress and nutrient deficiency. Microorganisms applied directly are easily lost and die in complex and harsh soil environments, resulting in unstable remediation effects.

[0007] In summary, existing technologies lack a comprehensive solution that simultaneously possesses the following characteristics: first, a Bacillus belyssus strain with broad-spectrum and high tolerance to multiple high-concentration heavy metals; second, a carrier and preparation process that can effectively immobilize the bacterial cells, protect their activity, and specifically improve the physicochemical properties of the soil; and third, a synergistic remediation method that can simultaneously achieve heavy metal solidification, soil fertility enhancement, and ecological function restoration.

[0008] Therefore, there is an urgent need in this field to screen out functional strains with stronger multi-resistance and develop efficient and stable microbial remediation agents and application methods to overcome the shortcomings of existing technologies and achieve efficient, low-cost and ecologically sustainable remediation of contaminated soil in mining areas. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a heavy metal-resistant Bacillus berghei and its microbial remediation agent and application. Heavy metal-resistant Bacillus berghei is obtained by screening soil from heavy metal-contaminated mining areas. Utilizing the unique physiological and biochemical characteristics of Bacillus berghei, a microbial remediation agent for heavy metal-contaminated soil remediation is prepared, achieving efficient and low-cost remediation of heavy metal-contaminated mining area soil and improving the ecological function of heavy metal-contaminated soil.

[0010] One objective of this invention is to provide a strain of Bacillus velezensis DBS01, deposited at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China), with accession number CCTCC NO: M 20252228 and deposit date of October 16, 2025. This strain was isolated from heavy metal-contaminated soil in the Dabao Mountain mining area of ​​Shaoguan City, Guangdong Province, and obtained through multi-step gradient heavy metal acclimatization. It exhibits significantly higher tolerance to multiple heavy metals such as Cu, Pb, Zn, Cd, As, and Hg than ordinary strains (e.g., in soil containing 1000 mg / L Cu). 2+ Or 1500 mg / L Pb 2+ It can still grow in the culture medium.

[0011] The screening process for Bacillus velezensis DBS01 is as follows:

[0012] (1) Select the Dabao Mountain tailings area, where the tailings have been piled up for more than 1 year;

[0013] (2) Using the multi-point mixed sampling method, collect 5-10 sampling points in the 0cm-20cm surface tailings area, mix them, remove larger stones, put them into sterile sampling bags, and refrigerate at 2℃-6℃.

[0014] (3) Weigh the tailings soil according to the ratio of 5g-15g of tailings soil to 80mL-120mL of physiological saline, add sterile physiological saline, shake at 160r / min-200r / min for 20min-40min at room temperature, let stand for 10min-20min and take the supernatant as bacterial suspension.

[0015] (4) After the bacterial suspension was serially diluted 0, 10, 100, 1000 and 10000 times, it was inoculated into beef extract peptone medium (typical medium formula: 1L medium contains 3g beef extract, 10g peptone, 5g NaCl and 20g agar).

[0016] (5) Incubate at 25℃ for 1-7 days;

[0017] (6) Primary acclimatization: Select single colonies from the culture medium and inoculate them with a concentration of 40 mg / L-70 mg / L Cu. 2+ In beef extract peptone liquid medium, incubate at 25°C for 1-5 days;

[0018] (7) Gradient stress acclimatization: Take the culture and inoculate it sequentially into Cu 2+The strain was cultured in liquid medium with concentrations increased stepwise from 100 mg / L to 1000 mg / L; this step was crucial in that it directed the screening and induced high-level heavy metal resistance / tolerance mechanisms in the strain by gradually increasing the stress pressure of a single heavy metal (Cu).

[0019] (8) Composite stress screening: The Cu samples subjected to the above-mentioned stress were screened. 2+ The acclimatized bacterial culture was inoculated with a solution containing 1500 mg / L Pb. 2+ 500mg / L Zn 2+ 100mg / L Cd 2+ 50mg / L As 3+ and 50 mg / L Hg 2+ The strain was cultured in a complex heavy metal culture medium; this step simulated the real complex pollution environment of the mining area, and finally screened out the target strain DBS01, which has extremely strong synergistic resistance to multiple heavy metals.

[0020] (9) The microorganisms were inoculated from the liquid culture medium onto beef extract peptone solid culture medium by streak inoculation to purify the microorganisms.

[0021] (10) The purified microorganism was identified as Bacillus velezensis by physiological and biochemical methods and was preserved.

[0022] The innovation of this strain lies in its extraordinary spectrum of resistance to multiple heavy metals, which stems from its isolation from the source of pollution and the aforementioned rigorous targeted acclimatization process, making it particularly suitable for the remediation of complex mining soils.

[0023] Another object of the present invention is to provide a microbial remediation agent comprising the above-mentioned Bacillus berleis DBS01 and a modified plant straw carrier (preferably aquatic plant straw).

[0024] Furthermore, the plant straw carrier is Fe 3+ Straw after soaking in solution and treatment with hydrogen peroxide solution.

[0025] Furthermore, the straw is selected from one or more of lotus pods, water hyacinth, Vallisneria natans, and reeds. Straw primarily originates from aquatic vegetation and accumulates elements such as phosphorus, potassium, and magnesium during its growth. These elements can provide nutrients for the restoration of mine soil function. Bacillus velezensis DBS01 adheres to the straw surface, and the organic acids it secretes can promote the release of organically bound and inorganic phosphorus from the straw. During phosphorus dissolution, phosphate ions can also combine with heavy metals to form precipitates, which is beneficial for the solidification of heavy metals in the soil.

[0026] The preparation method of the above-mentioned microbial remediation agent includes the following steps:

[0027] S1. Crush the plant straw and process it successively in Fe 3+ Modified straw was obtained by soaking in a solution and a hydrogen peroxide solution.

[0028] S2. Add the modified straw obtained in step S1 to the liquid culture medium, sterilize, and inoculate with Bacillus belye DBS01, then culture for 1-3 days; during this stage, the porous structure of the modified straw and Fe... 3+ Due to electrostatic adsorption, the strains are efficiently colonized inside and on the surface of the carrier.

[0029] S3. Add superphosphate to the culture system of step S2 and continue to culture for 3-5 days;

[0030] S4. Add agar and sodium alginate to the culture system of step S3 and continue to culture for 1-3 days;

[0031] S5. Freeze-dry the mixture obtained in step S4 to obtain a solid remediation agent, namely a microbial remediation agent.

[0032] Further, in step S1, Fe 3+ The solution concentration was 0.1 mg / L-0.5 mg / L, and the treatment time was 24-28 hours; Fe 3+ Fe in solution 3+ The source is selected from sodium iron ethylenediaminetetraacetate (which also has chelating function), iron oxide (slow-release iron supply), or iron sulfate (high-efficiency loading). 3+ Its main functions include: providing iron needed during vegetation restoration; positively charged Fe 3+ It can form an electrostatic attraction with negatively charged microorganisms, which is conducive to the adhesion of microorganisms to straw and provides a live microbial load.

[0033] Further, in step S1, the mass fraction of the hydrogen peroxide solution is 1%-5%, and the treatment time is 12-24 hours. ·OH oxidation modification and Fe 3+ Modification Synergistic Effect: Fe 3+ It undergoes a Fenton-like reaction with H2O2 to generate highly reactive hydroxyl radicals (·OH), which can efficiently oxidize and degrade cellulose on the surface of straw, producing a large number of micropores and nanopores, and introducing abundant oxygen-containing functional groups such as hydroxyl and carboxyl groups. This not only greatly increases the specific surface area and heavy metal adsorption sites of straw, but its rough and porous surface is also more conducive to the anchoring and hiding of microorganisms.

[0034] Furthermore, in step S3, the amount of superphosphate added is 1%-5% of the total mass of the culture system. This stage is the phosphorus dissolution and aggregation stage. Strains in the logarithmic growth phase secrete large amounts of organic acids such as citric acid and oxalic acid, which dissolve the superphosphate and release plant-available phosphorus and calcium. 2+ On the other hand, Ca 2+ As a cation bridge, it enhances the cross-linking and aggregation between negatively charged bacterial cells and the carrier, forming a more stable micro-ecosystem.

[0035] Further, in step S4, the amount of agar added is 0.1%-0.5% of the total mass of the culture system; the amount of sodium alginate added is 0.1%-0.5% of the total mass of the culture system. This stage is the immobilization and shaping stage, where sodium alginate is added to Ca... 2+ In the presence of agar, an ionogel reaction occurs, forming a three-dimensional network gel that encapsulates and fixes the straw carrier colonized with highly active bacteria, effectively protecting the activity of the bacteria in dry and soil environments.

[0036] Furthermore, in step S5, the viable count of Bacillus vesiculosus DBS01 in the microbial remediation agent can be stably maintained at no less than 0.5 × 10⁻⁶. 6 CFU / g, preferably up to 10 8 CFU / g-10 10 On the order of CFU / g.

[0037] The aforementioned microbial remediation agent is used to remediate heavy metal contaminated soil, which is mining soil contaminated with one or more of Cd, Cu, Pb, Zn, As, and Hg; the application rate of the microbial remediation agent is 20 g / m³. 2 -100 g / m 2 50g / m 2 -80 g / m 2 .

[0038] In its early growth stages, *Bacillus velezensis* DBS01 produces cellulase, providing nutrients for soil function restoration and vegetation growth. During its logarithmic growth phase, it secretes various organic acids such as citric acid and oxalic acid, promoting the dissolution of superphosphate and releasing phosphorus, further supporting soil function restoration and vegetation growth. In its stable growth phase, it secretes substances such as indoleacetic acid, antimicrobial peptides, and lipopeptides. Indoleacetic acid provides hormones for vegetation growth during the remediation process. Antimicrobial peptides and lipopeptides, containing basic amino acid residues, are alkaline and can significantly improve soil pH in mining areas, reducing the migration of heavy metals and facilitating their immobilization. Furthermore, these substances inhibit the growth and reproduction of acid-producing microorganisms in the soil (such as some fungi and sulfur-oxidizing bacteria). This reduces the production of organic acids and sulfuric acid by these microorganisms, slowing down soil acidification at its source.

[0039] The beneficial effects of this invention are as follows:

[0040] (1) Excellent solidification effect on multiple heavy metals: The strains and microbial remediation agents of this invention can effectively convert highly active acid-extractable heavy metals into stable residual states. Experiments have shown that after application, the acid-extractable state of Cu decreased from 62.1% to 9.5%, while the residual state increased from 26.8% to 79.6%. It also exhibits simultaneous and efficient solidification capabilities for Pb, Cd, Zn, As, and Hg, significantly reducing environmental risks.

[0041] (2) Simultaneous improvement of soil physicochemical properties: The microbial remediation agent of the present invention can simultaneously neutralize soil acidity and improve fertility. After application, the soil pH improved from 4.1 to 7.2; at the same time, the total phosphorus, total nitrogen and total potassium contents were increased several times, creating the necessary conditions for ecological restoration.

[0042] (3) Highly promotes plant growth: The microbial remediation agent of this invention can effectively eliminate the toxicity of soil to plants. After remediation, the germination rate, root length and shoot length of mung beans on the soil all increased significantly, and the biomass of the subsequently planted vegetation also increased significantly, proving its ability to drive ecological restoration.

[0043] (4) High activity and stable environment of the microbial agent: Through a unique carrier immobilization process, the viable count of functional bacteria in the microbial remediation agent is ensured to be high (not less than 0.5 × 10⁻⁶). 6 CFU / g, up to 10 8 CFU / g-10 10 (CFU / g), which enables it to maintain high activity and long-lasting efficacy in harsh soils, ensuring stable and long-lasting remediation effects. Attached Figure Description

[0044] Figure 1 Bacillus velezensis DBS01 was tested in samples containing different concentrations of heavy metals (200 mg / L Cu). 2+ 200mg / LPb 2+ 200mg / L Zn 2+ Cd 2+ 50mg / L, 25mg / L As 3+ Hg 2+ Growth curve in liquid culture medium (25 mg / L).

[0045] Figure 2 The image shows the germination rate of mung beans after the microbial remediation agent of this invention was used to remediate contaminated soil in a mining area.

[0046] Figure 3 The image shows the test results of mung bean root and shoot lengths after the soil remediation agent of this invention was used to remediate contaminated soil in a mining area.

[0047] Figure 4 This is a diagram of the biological quality of the restored vegetation. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited thereto.

[0049] Example 1

[0050] Bacillus velezensis DBS01 was screened from heavy metal-contaminated soil in the Dabao Mountain mining area of ​​Shaoguan City, Guangdong Province. The specific screening process is as follows:

[0051] (1) Select the Dabao Mountain tailings area, where the tailings have been piled up for more than 1 year;

[0052] (2) Using a multi-point mixed sampling method, 10 sampling points were collected in the 0cm-20cm surface tailings area. After mixing, larger stones were removed, and the samples were placed in sterile sampling bags and refrigerated at 4℃.

[0053] (3) Weigh 10g of tailings soil, add 90mL of sterile physiological saline, shake at 180r / min for 30min at room temperature, let stand for 20min and take the supernatant as bacterial suspension.

[0054] (4) The bacterial suspension was serially diluted 0, 10, 100, 1000 and 10000 times and then inoculated into beef extract peptone medium (medium formula: 1L of medium contains 3g beef extract, 10g peptone, 5g NaCl and 20g agar).

[0055] (5) Incubate at 25℃ for 5 days;

[0056] (6) Select a single colony from the culture medium and inoculate it with a solution containing 50 mg / L Cu. 2+ In beef extract peptone liquid medium, at 25°C, at 180 r / min, cultured for 3 days;

[0057] (7) By successively increasing Cu 2+ Concentration is used to achieve microbial domestication. Take 1 mL of the cultured bacterial solution and inoculate it sequentially into a solution containing Cu. 2+ Liquid culture media with concentrations of 100 mg / L, 150 mg / L, 200 mg / L, 300 mg / L, 500 mg / L, 700 mg / L, and 1000 mg / L were inoculated at 25°C and 180 rpm, including media containing Cu. 2+ Cultured for 3 days in culture media with concentrations of 100 mg / L, 150 mg / L, and 200 mg / L, then inoculated with Cu 2+ Cultured for 5 days in culture media with concentrations of 500 mg / L, 700 mg / L, and 1000 mg / L.

[0058] (8) Take 1 mL containing Cu 2+ Bacterial suspensions grown at a concentration of 1000 mg / L were inoculated onto substrates containing different concentrations of heavy metals (including 1500 mg / L Pb). 2+ 500mg / L Zn 2+ Cd 2+ 100mg / L, 50mg / L As 3+ Hg 2+ In a liquid culture medium of 50 mg / L, at 25°C, at 180 r / min, culture for 3 days;

[0059] (9) Microorganisms resistant to heavy metals were purified by inoculating them from liquid culture medium onto beef extract peptone solid culture medium using the streak inoculation method.

[0060] (10) The purified microorganism was identified as Bacillus velezensis by physiological and biochemical methods and named Bacillus velezensis DBS01. It is deposited at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), accession number: CCTCC M 20252228.

[0061] High concentrations of heavy metals in the soil of mining areas will prolong the time it takes for microorganisms to reach the logarithmic growth phase. For example... Figure 1As shown, when different concentrations of heavy metals were added to the culture medium during the cultivation of Bacillus velezensis DBS01, the time it took for Bacillus velezensis DBS01 to reach the logarithmic growth phase was prolonged.

[0062] Example 2

[0063] (1) Preparation of the repair agent: Lotus pods were selected as the plant straw. After drying, the lotus pods were crushed using a pulverizer. They were then soaked in sodium ferric ethylenediaminetetraacetate (EDTA) at a concentration of 0.2 mg / L for 24 hours. The adsorbed Fe was then removed. 3+ Lotus pods were transferred to a 5% hydrogen peroxide solution and treated for 24 hours, then freeze-dried. Beef extract peptone liquid culture medium was prepared, and the freeze-dried lotus pods were added to the medium at a concentration of 0.1 g / L. The medium was then steam-sterilized at 121°C for 15 minutes and cooled for later use. 0.5 mL of a bacterial suspension containing *Bacillus velezensis* DBS01 was inoculated and cultured at 25°C for 3 days. 3% (by weight) of superphosphate was added to the culture system, and the culture was continued for 5 days. Sterilized agar and sodium alginate were then added, at 0.2% and 0.5% (by weight) of the total culture system, respectively. The culture was continued for 3 days, and the mixture containing *Bacillus velezensis* DBS01 was freeze-dried to obtain the microbial remediation agent, in which the viable count of *Bacillus velezensis* DBS01 was 1.5 × 10⁻⁶. 10 CFU / g.

[0064] (2) Soil remediation experiment: The above-mentioned microbial remediation agent was mixed with the soil in the mining area at a ratio of 0%, 1%, 3% and 5%, respectively, and cultured for 30-60 days at a temperature of 25℃ and a humidity of 75%.

[0065] (3) Determination of heavy metal speciation in soil: 100g of remediated soil was taken and dried. The various heavy metal speciations in the soil were determined by the Tessier method and the BCR method. The soil nutrient status was also analyzed, as shown in Tables 1-7 below.

[0066] Table 1. Different forms of Cu in soil

[0067]

[0068] Table 2 Different forms of Pb in soil

[0069]

[0070] Table 3 Different forms of Cd in soil

[0071]

[0072] Table 4 Different forms of Zn in soil

[0073]

[0074] Table 5 Different forms of As in soil

[0075]

[0076] Table 6 Different forms of Hg in soil

[0077]

[0078] Table 7. Soil nutrient characteristics (element content in g / Kg)

[0079]

[0080] As shown in Tables 1 to 7, the microbial remediation agent provided by this invention exhibits excellent comprehensive remediation efficacy for soils contaminated with multiple heavy metals in mining areas. Regarding heavy metal solidification, the remediation agent significantly promotes the transformation of highly bioactive and toxic acid-extractable heavy metals into stable, low-migration residual forms. For the key pollutant Cu, its acid-extractable content decreased significantly from 62.1% to 9.5%, while the residual proportion increased significantly from 26.8% to 79.6% (as shown in Table 1). Similar stabilization trends were observed for other heavy metals such as Pb, Cd, Zn, As, and Hg (as shown in Tables 2 to 6), effectively reducing the overall environmental risk of complex pollution. In terms of soil ecological function restoration, the remediation agent simultaneously neutralized soil acidity and activated key nutrients, improving the soil pH from strongly acidic 4.1 to near-neutral 7.2, while the total phosphorus, total nitrogen, and total potassium contents of the soil were all increased several times (as shown in Table 7). The above data fully demonstrate the dual core effects of the microbial remediation agent of this invention in "solidifying heavy metals" and "improving soil", laying a solid foundation for the ecological reconstruction of mining area soil.

[0081] Example 3

[0082] (1) Preparation of repair agent: Lotus seedpods and water hyacinth were selected as plant straws. After drying, the lotus seedpods and water hyacinth were crushed using a pulverizer. Iron oxide was added, and the mass content of iron oxide in the mixture was 1%. The mixture was mixed for 24 hours and then transferred to a 1% hydrogen peroxide solution for 24 hours. After drying, beef extract peptone liquid culture medium was prepared. The dried straw mixture was added to the beef extract peptone liquid culture medium at a rate of 0.1 g / L of liquid culture medium. The mixture was steam sterilized at 121℃ for 15 minutes and then cooled for later use. Inoculate 1 mL of bacterial suspension containing Bacillus velezensis DBS01 and incubate at 25°C for 3 days. Add 5% (by weight) of superphosphate to the culture system and continue incubating for another 3 days. Add sterilized agar and sodium alginate, with agar and sodium alginate accounting for 0.5% and 0.5% of the total culture system mass, respectively. Continue incubating for another 3 days. Freeze-dry the mixture containing Bacillus velezensis DBS01 to obtain the microbial remediation agent, in which the viable count of Bacillus velezensis DBS01 is 5 × 10⁻⁶. 9 CFU / g.

[0083] (2) Soil remediation experiment: The above mixture was mixed with the soil in the mining area at a ratio of 0%, 1%, 3% and 5%, respectively. The application rates were 0 g / m², 20 g / m², 40 g / m² and 75 g / m², respectively. The physicochemical properties of the soil were measured at 0 days, 30 days, 90 days and 180 days (see Tables 8-11 below).

[0084] Table 8 Changes in the physicochemical properties of soil without added remediation agents (elemental content in g / Kg)

[0085]

[0086] Table 9 Changes in soil physicochemical properties after adding 1% remediation agent (element content in g / Kg)

[0087]

[0088] Table 10 Changes in soil physicochemical properties after adding 3% remediation agent (element content in g / Kg)

[0089]

[0090] Table 11 Changes in soil physicochemical properties after adding 5% remediation agent (element content in g / Kg)

[0091]

[0092] Tables 8 to 11 dynamically reveal the continuous improvement effect of the microbial remediation agent provided by this invention on the physical and chemical properties of the soil at different incubation times. Compared with the control group without the remediation agent (as shown in Table 8), where various indicators remained at a poor level for a long time, the soil pH value increased significantly with the incubation time after the application of the remediation agent, rapidly improving from strongly acidic (~4.2) to neutral (~7.1), effectively reversing the soil acidification process. At the same time, key fertility indicators such as soil organic matter, total phosphorus, and total nitrogen all showed a stable and significant increasing trend with the remediation time (as shown in Tables 9-11). After 180 days of treatment with 3% remediation agent, soil organic matter increased from 7.23 g / kg to 17.43 g / kg, and total phosphorus increased from 0.22 g / kg to 1.53 g / kg (as shown in Table 10). These data fully demonstrate that the microbial remediation agent of this invention not only takes effect quickly but also has a long-lasting and stable improvement ability, continuously injecting power into the restoration of the soil ecosystem and providing a reliable guarantee for the long-term ecological reconstruction of mining areas.

[0093] Example 4

[0094] (1) Preparation of repair agent: The plant straw is selected from Vallisneria natans. After drying, Vallisneria natans is crushed using a pulverizer. Ferric sulfate and sodium ferric ethylenediaminetetraacetate with a concentration of 0.1 mg / L are added and mixed for 24 h. The mixture is then transferred to a 5% hydrogen peroxide solution for 24 h and dried. Beef extract peptone liquid culture medium is prepared. The dried straw is added to the beef extract peptone liquid culture medium at a concentration of 0.1 g / L of liquid culture medium. The mixture is then steam sterilized at 121℃ for 15 min and cooled for later use. Inoculate 0.5 mL of bacterial culture containing Bacillus velezensis DBS01 and incubate at 25°C for 5 days. Add 5% (by weight) of superphosphate to the culture system and continue incubating for another 5 days. Add sterilized agar and sodium alginate, at 0.5% (by weight) and 0.5% (by weight) of the total culture system, respectively. Continue incubating for another 3 days. Freeze-dry the mixture containing Bacillus velezensis DBS01 to obtain the microbial remediation agent, in which the viable count of Bacillus velezensis DBS01 is 2 × 10⁻⁶. 10 CFU / g.

[0095] (2) Soil grouping: control group, pollution group and remediation group soil were set up. The control group was uncontaminated farmland soil, the pollution group was mining area soil without added remediation agent, the remediation group was mining area soil with added remediation agent, remediation group 1 was mining area contaminated soil with added 3% remediation agent, and remediation group 2 was mining area contaminated soil with added 5% remediation agent.

[0096] (3) Seed germination test: Soil samples were air-dried naturally, passed through a 2mm sieve to remove impurities such as stones and plant debris; each petri dish was lined with two layers of qualitative filter paper, and 50g of soil from the corresponding group was added. After leveling the surface, the dish was thoroughly watered with deionized water (soil moisture content was maintained at 65%-75%); 20 mung bean seeds were sown in each replicate of each group, evenly placed on the soil surface, and then covered with 1cm of the corresponding soil; all petri dishes were placed in a constant temperature incubator at 25℃ and cultured in the dark for 7 days; water was replenished with deionized water to maintain a stable soil humidity of 75%; germination potential was recorded from day 3 (number of germinated seeds / total number of seeds × 100%), and germination rate was recorded on day 7 (number of normally germinated seeds / total number of seeds × 100%) (e.g., Figure 2 As shown), measure the root length and shoot length of the germinating seed (e.g. Figure 3 (As shown in the image). After adding 3% and 5% (by mass) of the remediation agent, the germination rate of mung bean seeds significantly increased compared to the contaminated soil without the agent. On day 7, the germination rates increased from 25.6% to 90.6% and 92.1%, respectively, very close to the control group's germination rate (94.5%). Simultaneously, the addition of 3% and 5% of the remediation agent significantly increased the length of mung bean roots and shoots. The average root length increased from 1.34 cm to 3.15 cm and 3.26 cm, respectively, and the shoot length increased from 1.87 cm to 3.98 cm and 4.12 cm, respectively. This indicates that the remediation agent can not only mitigate the toxic effects of heavy metals in the soil on plants but also significantly promote plant growth and improve plant health.

[0097] Example 5

[0098] (1) Preparation of repair agent: The plant straw used is Vallisneria natans and Reed. After drying, Vallisneria natans and Reed are crushed using a pulverizer. Ferric sulfate and sodium ferric ethylenediaminetetraacetate with a concentration of 0.1 mg / L and iron oxide with a mass fraction of 1% are added. After mixing for 24 h, the mixture is transferred to a hydrogen peroxide solution with a mass fraction of 3% and treated for 12 h. After drying, beef extract peptone liquid culture medium is prepared. The dried mixture is added to the beef extract peptone liquid culture medium. The amount of straw mixture added is 0.1 g / L of liquid culture medium. The mixture is steam sterilized at 121℃ for 15 min and then cooled for later use. Inoculate 0.5 mL of bacterial culture containing Bacillus velezensis DBS01 and incubate at 25°C for 3 days. Add 2% (by weight) of superphosphate to the culture system and continue incubating for another 3 days. Add sterilized agar and sodium alginate, with agar and sodium alginate accounting for 0.1% and 0.5% (by weight) of the total culture system, respectively. Incubate for another 3 days and freeze-dry the mixture containing Bacillus velezensis DBS01, i.e., the microbial remediation agent, in which the viable count of Bacillus velezensis DBS01 is 5 × 10⁻⁶. 10 CFU / g.

[0099] (2) The heavy metal contaminated soil in the mining area was treated with remediation agents at dosages of 20 g / m², 50 g / m², and 100 g / m². After 90 days of treatment, ryegrass (Lolium perenne L.), bermudagrass (Cynodon dactylon (L.) Pers.), and centipede grass (Pteris vittata L.) were sown. The remediation agent was applied every 10 days, and water was sprayed regularly. After three consecutive applications, the remediation agent was applied every 20 days, and water was sprayed regularly. After three consecutive applications, the plants were harvested, dried, and weighed to determine the vegetation biomass per square meter. The results are as follows: Figure 4 As shown, increasing the amount of remediation agent significantly improved the dry weight of biomass after remediation. Under the condition of 100 g / m² application of remediation agent, the dry weight of biomass reached 223 g / m², indicating that the remediation agent can significantly improve soil quality, improve vegetation growth, and remediate polluted soil.

[0100] Comparative Example 1

[0101] The preparation of the repair agent is the same as in Example 4, except that it is Fe-free. 3+ Modification of straw.

[0102] The viable count of Bacillus vesiculus DBS01 in the obtained repair agent was 1×10⁻⁶. 7 CFU / g.

[0103] No Fe 3+ Modification of straw significantly reduces the colonization efficiency of microorganisms in the final remediation agent, leading to a substantial decrease in the number of viable bacteria. Furthermore, due to the lack of Fe... 3+ Due to the "bridging" effect and porous structure of the soil, the adsorption and solidification capacity of the remediation agent for heavy metals is weakened, and the soil remediation effect (such as pH increase and residual state conversion rate) is significantly worse.

[0104] Comparative Example 2

[0105] The preparation of the repair agent is the same as in Example 4, except that hydrogen peroxide is not used to modify the straw.

[0106] The viable count of Bacillus vesiculus DBS01 in the obtained repair agent was 2.5 × 10⁻⁶. 7 CFU / g.

[0107] Straw carrier only physically loads Fe 3+ It failed to produce a porous structure and abundant functional groups. Its adsorption capacity was limited, and the deterioration of the microbial colonization environment led to a significant decrease in the number of viable bacteria, and its repair effect also decreased accordingly.

[0108] Comparative Example 3

[0109] The preparation of the repair agent is the same as in Example 4, except that the straw is not treated with Fe. 3+Hydrogen peroxide treatment allows for direct use in the preparation of repair agents, even when using unmodified straw.

[0110] The viable count of Bacillus vesiculus DBS01 in the obtained repair agent was 0.5 × 10⁻⁶. 7 CFU / g.

[0111] Comparative Example 4

[0112] The preparation of the repair agent is the same as in Example 4, except that superphosphate is not used.

[0113] The viable count of Bacillus vesiculus DBS01 in the obtained repair agent was 0.5 × 10⁻⁶. 8 CFU / g.

[0114] The repair agent lacks key phosphorus and calcium sources. 2+ The strain's phosphorus-solubilizing and growth-promoting effects were not fully realized, resulting in a minimal increase in total soil phosphorus content; simultaneously, a lack of Ca... 2+ Due to the cross-linking effect, the immobilization effect of sodium alginate deteriorates, which may lead to an increase in the mortality rate of bacteria during the drying process, a decrease in the number of viable bacteria, and poor structure of the repair agent.

[0115] Comparative Example 5

[0116] The preparation of the repair agent is the same as in Example 4, except that: the Bacillus belye mentioned in the background art CN120758429A is used, that is, the existing technology strain is used.

[0117] The viable count of Bacillus vesiculosus in the obtained repair agent was 3.0 × 10⁻⁶. 8 CFU / g.

[0118] The results indicate that, using the same preparation method, the strain DBS01 of this invention has a significant advantage in maintaining activity compared to existing strains.

[0119] Comparative Example 6

[0120] The bacterial solution, simply crushed straw (unmodified), superphosphate, etc. are physically mixed and then applied directly, which is the traditional physical mixing (non-immobilization) method.

[0121] The viable count of Bacillus belysinus DBS01 in the resulting mixture was 1 × 10⁻⁶. 6 CFU / g.

[0122] The results demonstrate the crucial role of the "multi-stage fermentation + immobilization" process of this invention in protecting cell activity and achieving long-term repair.

Claims

1. A heavy metal-resistant Bacillus belye ( Bacillus velezensis Its characteristics are, The Bacillus belyssus mentioned is Bacillus belyssus DBS01, which is deposited at the China Center for Type Culture Collection (CCTCCNO: M 20252228) on October 16, 2025.

2. A microbial remediation agent, characterized in that, It comprises Bacillus belye DBS01 as described in claim 1 and a modified plant straw carrier, wherein the modified plant straw carrier is Fe... 3+ Straw after soaking in solution and treatment with hydrogen peroxide solution.

3. The microbial remediation agent according to claim 2, characterized in that, The straw is selected from one or more of the following: lotus seedpods, water hyacinths, eelgrass, and reeds.

4. The microbial remediation agent according to claim 3, characterized in that, Fe 3+ Fe in solution 3+ The source is one or more of ferric sodium ethylenediaminetetraacetate, iron oxide, and ferric sulfate, Fe 3+ The concentration is 0.1 mg / L-0.5 mg / L; the mass fraction of the hydrogen peroxide solution is 1%-5%.

5. A method for preparing the microbial remediation agent as described in any one of claims 2 to 4, characterized in that, Includes the following steps: S1. Crush the plant straw and process it successively in Fe... 3+ Modified straw was obtained by soaking in a solution and a hydrogen peroxide solution. S2. Add the modified straw obtained in step S1 to the liquid culture medium, sterilize it, inoculate it with Bacillus belye DBS01, and culture for 1-3 days; S3. Add superphosphate to the culture system of step S2 and continue to culture for 3-5 days; S4. Add agar and sodium alginate to the culture system of step S3 and continue to culture for 1-3 days; S5. Freeze-dry the mixture obtained in step S4 to obtain a solid remediation agent, namely a microbial remediation agent.

6. The method according to claim 5, characterized in that, In step S3, the amount of superphosphate added is 1%-5% of the total mass of the culture system.

7. The method according to claim 5, characterized in that, In step S4, the amount of agar added is 0.1%-0.5% of the total mass of the culture system; the amount of sodium alginate added is 0.1%-0.5% of the total mass of the culture system.

8. The method according to claim 5, characterized in that, In step S5, the viable count of Bacillus vesiculus DBS01 in the microbial remediation agent is not less than 0.5 × 10⁻⁶. 6 CFU / g.

9. The application of a microbial remediation agent as described in any one of claims 2 to 4 in the remediation of heavy metal contaminated soil.

10. The application according to claim 9, characterized in that, The heavy metal contaminated soil is mining area soil contaminated with one or more of Cd, Cu, Pb, Zn, As, and Hg; the application rate of the microbial remediation agent is 20 g / m³. 2 -100 g / m 2 .

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