Compound microbial agent for treating mine tailing polluted soil as well as preparation method and application of compound microbial agent

Abstract

The invention belongs to the technical field of soil microbial control, and discloses a compound microbial agent for treating mine tailing polluted soil, the compound microbial agent comprises a coxsackie P1-4-containing bacterial liquid and a stenotrophomonas R5-containing bacterial liquid in a volume ratio of (1-2): (1-2), and the OD600 value of the coxsackie P1-4-containing bacterial liquid and the OD600 value of the stenotrophomonas R5-containing bacterial liquid are independently 0.5-1.5. The invention further discloses a preparation method of the complex microbial inoculant and application of the complex microbial inoculant in treatment of mine tailing polluted soil. The coxakia P1-4 and the stenotrophomonas R5 screened by the invention are both derived from a cadmium-resistant environment (rhizosphere of miscanthus sinensis and endogenous rice), and have natural tolerance to heavy metal stress; the microbial agent prepared by compounding the microbial agent shows extremely strong adaptability and repair pertinence to typical acidic and cadmium-lead combined polluted soil in mine tailing areas, and solves the general problems of low survival rate and function failure of a common microbial agent in a severe tailing environment.

Description

Technical Field

[0001] This invention belongs to the field of soil microbial control technology, and particularly relates to a compound microbial agent for treating soil contaminated by mine tailings, its preparation method and application. Background Technology

[0002] Tailings generated during mining operations contain large amounts of heavy metals (such as cadmium and lead) and other harmful substances, causing serious pollution to the ecological environment. Traditionally, the treatment of this type of pollution has relied primarily on physical and chemical methods, such as: Physical methods include topsoil replacement, soil replacement, and isolation landfill. While these methods can produce quick results, they involve large-scale engineering projects and are costly. They are only suitable for small-scale pollution and do not fundamentally eliminate pollutants, posing risks of secondary treatment and pollution transfer.

[0003] Chemical methods, such as chemical fixation / stabilization, leaching, and redox reactions, alter the form or solubility of heavy metals by adding chemical reagents. However, these methods also suffer from high treatment costs, potential damage to soil structure, the introduction of new chemical substances causing secondary pollution, and uncertain long-term stability.

[0004] Given the limitations of traditional methods, bioremediation using microorganisms has attracted widespread attention in recent years due to its advantages such as environmental friendliness, relatively low cost, mild remediation process, and ecological sustainability. However, despite the promising prospects of microbial remediation technology, its practical application in complex tailings-contaminated soils is limited by the characteristics of the microorganisms and the complexity of the tailings environment. Most microbial remediation efforts have limited effectiveness and low efficiency. Therefore, developing a microbial agent to improve remediation outcomes is particularly important. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a composite microbial agent for treating soil contaminated by mine tailings, its preparation method and application.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows: A compound microbial agent for remediating soil contaminated by mine tailings, comprising Cossackella bacteria in a volume ratio of 1-2:1-2. (Kosakonia sacchari) Bacterial suspensions of P1-4 and containing oligotrophomonas ( Stenotrophomonas The bacterial suspension of sp.)R5 contained Coccidia. (Kosakonia sacchari) Bacterial suspensions of P1-4 and containing oligotrophomonas ( Stenotrophomonas The OD600 values ​​of the bacterial culture of sp.)R5 were independently 0.5~1.5; Among them, Cossackella (Kosakonia sacchari)P1-4 is deposited at the China Center for Type Culture Collection (CCTCC) on September 20, 2023, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20231759. Oligotrophomonas ( Stenotrophomonas sp.)R5 is deposited at the China Center for Type Culture Collection (CCTCC) on September 20, 2023, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20231758.

[0007] Based on a general inventive concept, the present invention also provides a method for preparing a compound microbial agent for treating mine tailings contaminated soil, comprising the following steps: (1) Cossackella (Kosakonia sacchari) P1-4 was inoculated into LB medium, shaken, and cultured. After culture, the supernatant was removed by centrifugation, and the OD600 value of the bacterial cells was adjusted to 0.5-1.5 to obtain Cossackella bacteria. (Kosakonia sugar ) Bacterial solution of P1-4; (2) Oligotrophomonas ( Stenotrophomonas sp.) R5 was inoculated into LB medium, shaken, and after culture, the supernatant was removed by centrifugation. The OD600 value of the bacterial cells was adjusted to 0.5-1.5 to obtain the oligotrophomonads (sp.) Stenotrophomonas bacterial culture of sp.)R5; (3) Containing Cossack bacteria (Kosakonia sacchari) Bacterial suspensions of P1-4 and containing oligotrophomonas ( Stenotrophomonas The bacterial solution of sp.)R5 was mixed in a volume ratio of 1~2:1~2 to obtain the composite microbial agent.

[0008] In the above preparation method, preferably, in step (1), the shaking culture conditions include a culture temperature of 26℃~30℃, a culture time of 10h~14h, and a rotation speed of 180rpm~220rpm. The shaking culture temperature is further preferably 28℃, the shaking culture time is further preferably 12h, and the OD600 value is further preferably 1.0.

[0009] In the above preparation method, preferably, in step (1), the centrifugation speed is 5800 rpm to 6200 rpm and the centrifugation time is 2 min to 4 min.

[0010] In the above preparation method, preferably, in step (2), the shaking culture conditions include a culture temperature of 26℃~30℃, a culture time of 10h~14h, and a rotation speed of 180rpm~220rpm. The shaking culture temperature is further preferably 28℃, the shaking culture time is further preferably 12h, and the OD600 value is further preferably 1.0.

[0011] In the above preparation method, preferably, in step (2), the centrifugation speed is 5800 rpm to 6200 rpm and the centrifugation time is 2 min to 4 min.

[0012] Based on a general inventive concept, the present invention also provides the application of the composite microbial agent as described above, or the composite microbial agent prepared by the above preparation method, in the remediation of mine tailings contaminated soil.

[0013] In the above-mentioned applications, preferably, 8-12L of compound microbial agent is applied to each cubic meter of mine tailings contaminated soil.

[0014] In the above-mentioned applications, preferably, the mine tailings contaminated soil mainly refers to soil contaminated with cadmium and / or lead.

[0015] In the above-mentioned application, preferably, the pH value of the mine tailings contaminated soil is 4-4.5, the available cadmium content is 0.5-0.7 mg / kg, the available lead content is 7-9 mg / kg, and the organic matter content is 5-7 mg / kg.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The Cossackella strains screened in this invention (Kosakonia sacchari) P1-4 and Oligotrophomonas ( Stenotrophomonas All of the sp.)R5 are derived from cadmium-tolerant environments (rhizosphere of Miscanthus sinensis and endophytic rice), and have natural tolerance to heavy metal stress. The bacterial agent made by compounding them shows strong adaptability and remediation targeting for typical acidic, cadmium- and lead-contaminated soils in mine tailings areas, solving the common problem of low survival rate and functional failure of ordinary microbial agents in harsh tailings environments.

[0017] (2) This invention is the first to use the strain Cossackella ( Kosakonia sacchari P1-4 and Oligotrophomonas ( Stenotrophomonas When combined with R5, the compound has a significant immobilization effect on cadmium (Cd) and lead (Pb) in mine tailings contaminated soil. Furthermore, the compounded bacterial agent can more effectively increase the pH value of contaminated soil and increase the soil organic matter content, thereby fundamentally improving the soil micro-ecological environment and making it more conducive to long-term stable remediation.

[0018] (3) This invention is based entirely on the life activities of microorganisms (such as adsorption, accumulation, transformation, etc.) to achieve the fixation and passivation of heavy metals. The entire remediation process does not introduce any exogenous chemical agents, thus avoiding the risks of soil compaction, salinization, and secondary chemical pollution that may be caused by traditional chemical solidification methods. After remediation, the microorganisms can become part of the soil ecosystem, resulting in high ecological safety.

[0019] Biological Preservation Instructions The oligotrophomonas involved in this application ( Stenotrophomonas sp.)R5 was deposited at the China Center for Type Culture Collection on September 20, 2023, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20231758; This application involves Cossacella ( Kosakonia sacchari P1-4 were deposited on September 20, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M20231759. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a diagram showing the screening and culture results of the 15 bacteria selected by this invention. Detailed Implementation

[0022] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0023] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0024] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0025] The culture media and their formulations are described below: 1 / 2LB liquid culture medium: Weigh 5.0 g sodium chloride, 2.5 g yeast extract and 5.0 g tryptone, and bring the volume to 1000 mL with ultrapure water. Adjust the pH to 7.0, autoclave at 121°C for 30 minutes, and use after cooling.

[0026] LB liquid medium: Weigh 10.0 g sodium chloride, 5.0 g yeast extract and 10.0 g tryptone, and bring the volume to 1000 mL with ultrapure water. Adjust the pH to 7.0, sterilize by autoclaving at 121°C for 30 minutes, and use after cooling.

[0027] NB liquid culture medium: Weigh 10.0 g of glucose, 3.0 g of beef extract and 5.0 g of peptone, and make up to 1000 mL with ultrapure water. Adjust the pH to 7.0, dispense into Erlenmeyer flasks, seal and sterilize by autoclaving at 121°C for 30 minutes. Use after cooling.

[0028] 1 / 2LB solid medium: Weigh 5.0 g sodium chloride, 2.5 g yeast extract and 5.0 g tryptone, and bring the volume to 1000 mL with ultrapure water. Adjust the pH to 7.0, add 15 g agar, and autoclave at 121°C for 30 minutes. Cool to 50°C and pour into plates.

[0029] LB solid medium: Weigh 10.0 g sodium chloride, 5.0 g yeast extract, and 10.0 g tryptone, and bring the volume to 1000 mL with ultrapure water. Adjust the pH to 7.0, add 15 g agar, and autoclave at 121°C for 30 minutes. Cool to 50°C and pour into plates.

[0030] NB solid medium: Weigh 10.0 g glucose, 3.0 g beef extract and 5.0 g peptone, and bring the volume to 1000 mL with ultrapure water. Adjust the pH to 7.0, add 15 g agar, and autoclave at 121°C for 30 minutes. Cool to 50°C and pour into plates.

[0031] Example 1: This example demonstrates the isolation and identification of the bacterial strain.

[0032] 1. Cossackie (Kosakonia sacchari) Isolation and culture of P1-4 At the sewage outlet of the Xiangjiang River, select vigorous Miscanthus sinensis, pull it out along with its roots, shake off the loose soil on the surface of the roots, collect the soil tightly attached to the surface of the roots (rhizosphere soil), put it into a pre-prepared sterile bag, seal it, and quickly bring it back to the laboratory for storage in a 4°C refrigerator.

[0033] Weigh 5g of soil and place it in a 45mL sterile Erlenmeyer flask containing glass beads. Shake for 20 minutes to thoroughly mix the soil sample with the water. Then let it stand. Take 1mL of the supernatant and dilute it to 1×10⁻⁶. 4 1×10 5 and 1×10 6After dilution, 100 μL of the diluted supernatant was spread onto LB agar plates and incubated upside down in a constant temperature incubator (28℃). The growth of the strains on the plates was observed periodically. After 5 days of growth, strains with different morphological characteristics (color, size, shape, etc.) were selected from the plates using an inoculation loop and streaked onto LB agar plates. Nine endophytic bacteria were screened and named: P1-1, P1-2, P1-3, P1-4, P1-5, P1-6, P1-7, P1-8, and P1-9. After activation of the single bacteria, 1.5 mL of the bacterial solution was aliquoted into 2 mL centrifuge tubes at a bacterial suspension to sterile glycerol ratio of 1:1 and stored at -80℃ for later use.

[0034] 2. Oligotrophomonas ( Stenotrophomonas sp. Isolation and culture of ) Plump and healthy Huanghuazhan rice seeds from Zhouxi Town, Kaili City, Guizhou Province were selected. First, the seeds were soaked in 75% anhydrous ethanol for 10 minutes, then the ethanol was poured off, and the seeds were rinsed five times with sterile water. Next, they were soaked in 5% NaClO for 10 minutes, and then rinsed five times with sterile water. 100 μL of the sterile water from the final rinse was spread onto 1 / 2 LB solid culture medium and incubated in a constant temperature incubator for 5 days. The presence of bacterial growth on the medium was observed to ensure thorough sterilization of the rice seed surface.

[0035] Endophytic bacteria were isolated from rice seeds using 1 / 2 LB and NB liquid media. Surface-sterilized rice seeds were ground into powder in a sterile mortar with a small amount of liquid nitrogen. The rice seed powder was then inoculated into 250 mL of 1 / 2 LB and NB liquid media using a sterile weighing spoon. After 36 hours of incubation in a shaker (28 ℃, 180 rpm / min), 1 mL of the bacterial suspension was diluted to 1×10⁻⁶. 4 1×10 5 and 1×10 6 After multiplying, 100 μL of bacterial suspension was spread onto the corresponding solid culture medium and incubated upside down in a constant temperature incubator (28 ℃). The growth of the strains on the plates was observed periodically. After 5 days of growth, strains with different morphological characteristics were selected from the plates using an inoculation loop and streaked onto the corresponding solid culture medium. Six bacterial strains were screened and numbered R1, R2, R3, R4, R5, and R6. After single-strain activation, 1.5 mL of the bacterial suspension was mixed with sterile glycerol at a ratio of 1:1 and aliquoted into 2 mL centrifuge tubes and stored at -80 ℃ for later use.

[0036] Fifteen different bacterial species were isolated from the rhizosphere soil of *Miscanthus sinensis* and *Rhizophora huanghuazhanensis* seeds. These isolated bacteria were inoculated into LB liquid medium and cultured in a shaker (28℃, 180 rpm) for 36 hours. 100 μL of each bacterial suspension was then spread onto LB solid medium with a Cd concentration of 4 mmol / L and incubated upside down in a 28℃ incubator for further selection. Growth was observed after 7 days. The results of each bacterial selection culture are shown below. Figure 1 As shown.

[0037] Depend on Figure 1 It can be seen that P1-4 and R5 bacteria have relatively dense colonies on their surface, while other culture dishes have relatively few or no colonies. This indicates that P1-4 and R5 bacteria have a certain degree of cadmium resistance, and P1-4 and R5 bacteria were selected for further research.

[0038] R5 bacteria are round with neat edges, a hemispherical shape, a plump texture, a smooth and moist surface, and are milky white and mucous-like. They can be pulled into threads when picked up and are Gram-negative bacteria. P1-4 bacteria are generally light in color and exhibit a relatively dense distribution, with a large number of tiny colonies clustered together to form a relatively concentrated colony area. They are also Gram-negative bacteria.

[0039] The isolated R5 and P1-4 strains were inoculated into LB liquid medium for activation. After centrifugation using a high-speed refrigerated centrifuge, the bacterial cells were collected, and bacterial DNA was extracted using a bacterial DNA extraction kit according to the manufacturer's instructions. The amplified DNA was then sent to Hunan Saisiwei Biotechnology Co., Ltd. for sequencing and identification. The obtained bacterial gene sequence was compared online with the NCBI database to identify the species of endophytic bacteria.

[0040] The results showed that strain R5 was... Stenotrophomonas sp. (Oligotrophomonas), named Oligotrophomonas (Stenotrophomonas sp. ） R5 was deposited at the China Center for Type Culture Collection on September 20, 2023, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20231758.

[0041] Strain P1-4 is a strain of the genus *Cossacchari*, with the Latin name... Kosakonia sacchari It was named Cossackie ( Kosakonia sacchari P1-4 were deposited on September 20, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20231759.

[0042] The 16S rDNA sequence of P1-4 bacteria is shown in SEQ ID NO: 1, and the specific sequence is as follows:

[0043] Example 2: This embodiment is to verify the presence of oligotrophomonas (… Stenotrophomonas sp.) R5 strain and Cossackella ( Kosakonia sacchari Whether strains P1-4 can coexist stably under co-culture conditions.

[0044] Select oligotrophomonas separately ( Stenotrophomonas sp.) R5 strain and Cossackella ( Kosakonia sugar Single colonies of strain P1-4 were inoculated into 5 mL of LB liquid medium and cultured at 28°C with shaking at 180 rpm for 12 h to obtain R5 and P1-4 bacterial suspensions, respectively. The OD600 value of the bacterial suspensions was measured using a UV spectrophotometer and adjusted to OD600=0.05 with LB liquid medium for later use.

[0045] The experiment was set up with 3 groups, each with 3 biological replicates. Each replicate consisted of 50 mL of LB liquid medium. The specific groupings are as follows: Control group 1 (R5 single culture group): Inoculated with 5 mL of adjusted R5 bacterial suspension, inoculation amount 10% (v / v). Control group 2 (P1-4 single culture group): 5 mL of adjusted P1-4 bacterial suspension was inoculated, with an inoculation volume of 10% (v / v). Experimental group (R5+P1-4 co-culture group): 2.5 mL of adjusted R5 bacterial solution and 2.5 mL of adjusted P1-4 bacterial solution were inoculated respectively, with a total inoculation volume of 10% (v / v) and an inoculation ratio of 1:1.

[0046] The above three culture systems were placed in a constant temperature shaking incubator at 37℃ and 180r / min. Samples were taken at 0, 2, 4, 6, 8, 12, 24 and 48h of culture, with three replicates taken from each group at each time point.

[0047] Viable bacteria count: Samples were taken at each time point and serially diluted with sterile physiological saline (10⁻⁶). -1 ~10 -8 ), take 100 μL of the diluted solution and spread it evenly on LB solid medium. After incubation at 28℃ for 24 h, count the number of colonies (CFU) based on the morphological characteristics of R5 and P1-4 colonies, and calculate the number of viable bacteria (CFU / mL) per milliliter of sample. The results are shown in Table 1.

[0048] Table 1. Viable bacterial count (CFU / mL) in each treated sample

[0049] As shown in Table 1, the viable count of strain R5 in the co-culture group was 1.01 × 10⁻⁶ at 12 h. 8 CFU / mL; the viable count of strain P1-4 at 12 h was 1.00 × 10⁻⁶. 8 The CFU / mL levels all met the expected growth targets. The viable count of strain R5 in the co-culture group was 1.92 × 10⁻⁶ at 24 h. 8 CFU / mL, compared to the single culture group (1.85×10⁻⁶). 8 The CFU / mL count increased by 3.8%; the viable count of strain P1-4 at 24 h was 1.88 × 10⁻⁶. 8 CFU / mL, compared to the single culture group (1.82×10⁻⁶). 8 The CFU / mL level increased by 3.3%, demonstrating a synergistic growth effect during co-culture. At 48 hours, the viable counts of strains R5 and P1-4 in the co-culture group remained at 1.89 × 10⁻⁶. 8 CFU / mL and 1.85×10 8 The high level of CFU / mL without significant decline further demonstrates that strains R5 and P1-4 can coexist stably under co-culture conditions.

[0050] Example 3: A method for preparing a compound microbial agent includes the following steps: (1) The Cossackella strains screened in Example 1 (Kosakonia sacchari) P1-4 was inoculated into LB liquid medium and cultured at 28°C with shaking at 200 rpm for 12 h. Then, it was centrifuged at 6000 rpm for 3 min, the supernatant was removed, the bacterial cells were collected, resuspended in sterile water, and the OD600 value of the bacterial suspension was adjusted to 1.0 to obtain Cossackie bacteria. (Kosakonia sacchari) Bacterial solution of P1-4; (2) The oligotrophomonas screened in Example 1 ( Stenotrophomonas sp. R5 cells were inoculated into LB liquid medium and cultured at 28°C with shaking at 200 rpm for 12 h. Then, the cells were centrifuged at 6000 rpm for 3 min, the supernatant was removed, and the cells were collected. The cells were resuspended in sterile water, and the OD600 value of the suspension was adjusted to 1.0 to obtain oligotrophomonas. (Stenotrophomonas sp.) R5 bacterial culture.

[0051] (3) Contains Cossackella (Kosakonia sacchari) Bacterial suspensions of P1-4 and containing oligotrophomonas (Stenotrophomonas sp.) R5 bacterial solutions were mixed in volume ratios of 2:1, 1:1, and 1:2 to form compound microbial agents.

[0052] The three compound microbial agents prepared above, and those containing only Cossacchari, were then used. (Kosakonia sacchari) The bacterial culture of P1-4 (OD600 value 1.0) contained only oligotrophomonas. (Stenotrophomonas sp.) The bacterial solution of R5 (OD600 value of 1.0) was compared with that of mine tailings contaminated soil.

[0053] Experimental group: Control group (no treatment); Treatment group 1: treated with microbial agents containing single P1-4 bacterial solution; Treatment group 2: treated with microbial agents containing single R5 bacterial suspension; Treatment group 3: Treatment with a microbial agent containing P1-4 bacterial solution and R5 bacterial solution in a volume ratio of 2:1; Treatment group 4: Treatment with a microbial agent containing P1-4 bacterial solution and R5 bacterial solution in a volume ratio of 1:1; Treatment group 5: The microbial agent was used to treat the P1-4 bacterial solution and R5 bacterial solution in a volume ratio of 1:2.

[0054] Experimental Methods: Contaminated soil from mine tailings was selected. Microbial agents from different treatment groups were added to the contaminated soil at a rate of 10 L per cubic meter of contaminated soil tailings. The mixture was stirred thoroughly. After 30 days, samples were taken to test the soil pH, available cadmium content, available lead content, and organic matter content. Available cadmium and lead content were tested according to the methods specified in "Determination of Eight Available Elements in Soil: Diethylenetriaminepentaacetic Acid Extraction-Inductively Coupled Plasma Atomic Emission Spectrometry (HJ 804-2016)". Organic matter content was tested according to the methods specified in "NY / T 1121.6-2006 Soil Testing Part 6: Determination of Soil Organic Matter". The test results are shown in Table 2.

[0055] Table 2 Soil pH, available cadmium content, available lead content, and organic matter content in different treatment groups

[0056] As shown in Table 2, both single-component P1-4 and single-component R5 microbial agents can effectively treat mine tailings contaminated soil. They can fix cadmium and lead in the mine tailings contaminated soil, increase the environmental pH, and remediate the mine tailings contaminated soil. The effect of the microbial agent after combining P1-4 and R5 microbial agents is significantly better than that of single-component agents. The combined microbial agent has a very high fixation efficiency for cadmium and lead in the mine tailings contaminated soil and can stably increase the environmental pH, which helps to rapidly remediate the mine tailings contaminated soil. Furthermore, the effect of treating mine tailings contaminated soil varies depending on the volume ratio of P1-4 and R5 microbial agents. The microbial agent obtained by combining P1-4 and R5 microbial agents in a volume ratio of 2:1 has the best treatment effect on mine tailings contaminated soil.

[0057] As can be seen from the above embodiments, the present invention screened and identified bacteria that can grow normally in cadmium environments, namely Cossackie bacillus P1-4 and oligotrophomonas R5. Microbial agents were prepared based on the bacterial solutions of these two bacteria for the remediation of mine tailings soil. Experiments showed that both the single P1-4 and single R5 bacterial solutions of the microbial agent can effectively treat mine tailings contaminated soil, fix cadmium and lead in the contaminated soil, and increase the environmental pH, thus remediating the contaminated soil. The microbial agent prepared by combining P1-4 and R5 bacterial solutions was more effective than the single-solution microbial agent. The combined microbial agent had extremely high fixation efficiency for cadmium and lead in mine tailings contaminated soil and could stably increase the environmental pH, which is conducive to the rapid remediation of mine tailings contaminated soil. The microbial agent prepared by combining P1-4 and R5 bacterial solutions in a volume ratio of 2:1 showed the best effect, providing a new solution for mine tailings contaminated soil.

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound microbial agent for treating soil contaminated by mine tailings, characterized in that, Including Cossac bacteria in a volume ratio of 1~2:1~2 (Kosakonia sacchari) Bacterial suspensions of P1-4 and containing oligotrophomonas ( Stenotrophomonas sp. The bacterial suspension of R5 contained Coccidia. (Kosakonia sacchari) Bacterial suspensions of P1-4 and containing oligotrophomonas ( Stenotrophomonas The OD600 values ​​of the bacterial culture of sp.)R5 were independently 0.5~1.5; Among them, Cossackella (Kosakonia sacchari) P1-4 is deposited at the China Center for Type Culture Collection (CCTCC) on September 20, 2023, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20231759. Oligotrophomonas ( Stenotrophomonas sp.)R5 is deposited at the China Center for Type Culture Collection (CCTCC) on September 20, 2023, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20231758.

2. A method for preparing a composite microbial agent for treating mine tailings contaminated soil, characterized in that, Includes the following steps: (1) Cossackella (Kosakonia sacchari) P1-4 was inoculated into LB medium, shaken, and cultured. After culture, the supernatant was removed by centrifugation, and the OD600 value of the bacterial cells was adjusted to 0.5-1.5 to obtain Cossackella bacteria. (Kosakonia sacchari ) Bacterial solution of P1-4; (2) Oligotrophomonas ( Stenotrophomonas sp.) R5 was inoculated into LB medium, shaken, and after culture, the supernatant was removed by centrifugation. The OD600 value of the bacterial cells was adjusted to 0.5-1.5 to obtain the oligotrophomonads (sp.) Stenotrophomonas bacterial culture of sp.)R5; (3) Containing Cossack bacteria (Kosakonia sacchari) Bacterial suspensions of P1-4 and containing oligotrophomonas ( Stenotrophomonas The bacterial solution of sp.)R5 was mixed at a volume ratio of 1~2:1~2 to obtain the composite microbial agent.

3. The preparation method according to claim 2, characterized in that, In step (1), the conditions for shaking culture include a culture temperature of 26℃~30℃, a culture time of 10h~14h, and a rotation speed of 180rpm~220rpm.

4. The preparation method according to claim 2, characterized in that, In step (1), the centrifugation speed is 5800 rpm to 6200 rpm and the centrifugation time is 2 min to 4 min.

5. The preparation method according to claim 2, characterized in that, In step (2), the conditions for shaking culture include a culture temperature of 26℃~30℃, a culture time of 10h~14h, and a rotation speed of 180rpm~220rpm.

6. The preparation method according to claim 2, characterized in that, In step (2), the centrifugation speed is 5800 rpm to 6200 rpm and the centrifugation time is 2 min to 4 min.

7. The application of a composite microbial agent as described in claim 1 or a composite microbial agent prepared by any one of claims 2 to 6 in the remediation of mine tailings contaminated soil.

8. The application as described in claim 7, characterized in that, Apply 8-12L of compound microbial agent to each cubic meter of mine tailings contaminated soil.

9. The application as described in claim 7, characterized in that, The mine tailings contaminated soil mainly refers to soil contaminated with cadmium and / or lead.

10. The application as described in claim 7, characterized in that, The soil contaminated by the mine tailings has a pH of 4-4.5, an available cadmium content of 0.5-0.7 mg / kg, an available lead content of 7-9 mg / kg, and an organic matter content of 5-7 mg / kg.

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