A cadmium-resistant composite microbial agent, a preparation method and application thereof

Abstract

This invention discloses an anti-cadmium compound microbial agent, its preparation method, and its application, belonging to the field of microbial technology. The anti-cadmium compound microbial agent comprises three microorganisms: Bacillus, Priestella megaterium, and Burkholderia spp. These three microorganisms do not exhibit antagonistic interactions and each can thrive in environments containing Cd. 2+ This invention also discloses a method for preparing the cadmium-resistant compound microbial agent, which includes activating and expanding the cultivation of three microorganisms to obtain seed liquid, and then mixing them in a certain proportion. This invention further discloses the application of this cadmium-resistant compound microbial agent in remediating cadmium-contaminated soil, reducing the content of available cadmium in the soil, fixing or transforming cadmium ions in the environment, and promoting plant growth in cadmium-contaminated environments. The cadmium-resistant compound microbial agent of this invention, through the synergistic metabolism and biosorption of the three microorganisms, can efficiently fix and transform available cadmium in the soil, and has the advantages of high remediation efficiency, environmental friendliness, and low cost.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to an anti-cadmium compound bacterial agent, its preparation method, and its application. Background Technology

[0002] Cadmium (Cd) is one of the most biotoxic heavy metals, characterized by its high mobility, easy absorption by crops, and ability to harm human health through the food chain. In recent years, due to activities such as industrial wastewater irrigation, excessive application of chemical fertilizers and pesticides, and mining, large areas of farmland soil in my country have faced varying degrees of cadmium pollution. Once in the soil, cadmium mainly exists in its bioavailable form, easily absorbed by plant roots and transported to the above-ground parts, leading to excessive cadmium content in agricultural products, seriously threatening food safety and human health.

[0003] Currently, remediation technologies for cadmium-contaminated soil mainly include physical remediation (topsoil replacement, electrodynamic remediation), chemical remediation (leaching, passivation), and bioremediation (phytoremediation, microbial remediation). Among these, microbial remediation has received widespread attention due to its advantages such as low cost, environmental friendliness, and non-destructive effect on soil structure. Utilizing cadmium-resistant microorganisms, through mechanisms such as biosorption, extracellular precipitation, intracellular accumulation, and enzymatic transformation, highly active available cadmium in the soil can be converted into a less active, stable form, thereby reducing the bioavailability and environmental risk of cadmium.

[0004] There are some reports on cadmium-resistant single strains or compound bacterial agents in the existing technology, but they generally have the following problems: (1) The single strain has limited cadmium resistance and low remediation efficiency; (2) There may be antagonistic effects between different strains in the compound microbial agent, resulting in the actual effect being lower than expected; (3) Most microbial agents rely on a single adsorption mechanism, and their cadmium fixation and transformation efficiency in complex soil environments is unstable. Summary of the Invention

[0005] To address the above problems, this invention provides an anti-cadmium compound microbial agent, its preparation method, and its application. This invention is achieved through the following technical solutions.

[0006] A cadmium-resistant compound microbial agent comprising three microorganisms: Bacillus sp., Priestia megaterium, and Burkholderia cepacia. These three microorganisms do not exhibit antagonistic interactions and each can thrive in environments containing Cd. 2+ It grows in a suitable environment.

[0007] As a further aspect of the present invention, the ratio of viable Bacillus, Priestella megaterium, and Burkholderia in the compound bacterial agent is (1-3):(0.5-2):(0.5-1.5).

[0008] As a further aspect of the present invention, the ratio of viable Bacillus, Priestella megaterium, and Burkholderia is 1:1:1.

[0009] A method for preparing an anti-cadmium compound bacterial agent includes the following steps: S1, Bacillus, Priestella megaterium and Burkholderia were activated and cultured to obtain their respective seed solutions;

[0010] S2, by mixing the seed liquids of Bacillus, Priestella megaterium and Burkholderia in a certain proportion, an anti-cadmium compound bacterial agent can be obtained.

[0011] As a further embodiment of the present invention, in step S1, the activation method is to inoculate Bacillus, Priestella megaterium and Burkholderia into LB medium and incubate them at 30°C for 24 hours.

[0012] As a further embodiment of the present invention, the method further includes the step of adding excipients, wherein in step S2, excipients are added simultaneously for mixing.

[0013] As a further aspect of the present invention, the excipient is one or more of a protective agent, a carrier, a dispersant, and a nutrient.

[0014] As a further embodiment of the present invention, the protective agent is glycerol or trehalose; the carrier is diatomaceous earth or wheat bran; the dispersant is sodium carboxymethyl cellulose; and the nutrient is glucose or peptone.

[0015] Application of a cadmium-resistant compound microbial agent, wherein the compound microbial agent is used in at least one of the following aspects: (a) Remediation of cadmium-contaminated soil; (b) Reduce the content of available cadmium in the soil; (c) Fixing or transforming cadmium ions in the environment; (d) Promote plant growth in cadmium-polluted environments.

[0016] As a further aspect of the present invention, its application method is as follows: The cadmium-resistant compound microbial agent was applied to cadmium-contaminated soil at a dosage of 210 g / L. 9 ~110 11 CFU / kg soil; The cadmium-resistant compound microbial agent is applied once before crop sowing, and the crops include, but are not limited to, rice, wheat, corn, leafy vegetables and alfalfa.

[0017] The beneficial effects of this invention are as follows: 1. Synergistic effect: The three microorganisms have different anti-cadmium mechanisms (e.g., Bacillus has a strong extracellular adsorption capacity, Priestella megaterium can produce cadmium-binding proteins, and Burkholderia has the ability to accumulate and transform cadmium intracellularly). When the three are combined, through synergistic metabolism and bioadsorption, the fixation and transformation efficiency of available cadmium is significantly higher than that of any single strain.

[0018] 2. No mutual antagonism: Experimental verification shows that the three strains of this invention can coexist well under co-culture conditions without mutual inhibition, ensuring the stability of the compound bacterial agent and the reliability of its repair effect.

[0019] 3. Broad-spectrum adaptability: All three strains were isolated from cadmium-contaminated environments and have a strong tolerance to high concentrations of cadmium, making them suitable for the remediation of cadmium-contaminated soils with different levels of pollution.

[0020] 4. Environmentally friendly: All strains used are from the environment and do not produce secondary pollution. They can be used in combination with other remediation technologies (such as phytoremediation) and have good application prospects. Attached Figure Description

[0021] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Images showing the growth of Bacillus subtilis after inoculation into LB medium and cadmium-containing LB medium, respectively. Figure 2 Images of *Priestella megaterium* after inoculation into LB medium and cadmium-containing LB medium, respectively. Figure 3 Images of Burkholderia growth after inoculation into LB medium and cadmium-containing LB medium. Detailed Implementation

[0023] 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.

[0024] A cadmium-resistant compound microbial agent comprising three microorganisms: Bacillus sp., Priestia megaterium, and Burkholderia cepacia. These three microorganisms do not exhibit antagonistic interactions and each can thrive in environments containing Cd. 2+ It grows in a suitable environment.

[0025] Preferably, in the compound microbial agent, the ratio of viable bacteria of Bacillus, Priestella megaterium and Burkholderia is (1-3):(0.5-2):(0.5-1.5), specifically, the ratio of viable bacteria of Bacillus, Priestella megaterium and Burkholderia is 1:1:1.

[0026] A method for preparing an anti-cadmium compound bacterial agent includes the following steps: S1, Bacillus, Priestella megaterium, and Burkholderia were activated and cultured on a large scale to obtain their respective seed cultures. Preferably, the activation method involves inoculating Bacillus, Priestella megaterium, and Burkholderia into LB medium and culturing them at 30°C for 24 hours.

[0027] S2, by mixing the seed liquids of Bacillus, Priestella megaterium and Burkholderia in a certain proportion, an anti-cadmium compound bacterial agent can be obtained.

[0028] Preferably, the method further includes a step of adding excipients. In step S2, excipients are added simultaneously for mixing. Specifically, the excipients are one or more of a protective agent, a carrier, a dispersant, and a nutrient. Specifically, the protective agent is glycerol or trehalose; the carrier is diatomaceous earth or wheat bran; the dispersant is sodium carboxymethyl cellulose; and the nutrient is glucose or peptone.

[0029] Application of a cadmium-resistant compound microbial agent, wherein the compound microbial agent is used in at least one of the following aspects: (a) Remediation of cadmium-contaminated soil; (b) Reduce the content of available cadmium in the soil; (c) Fixing or transforming cadmium ions in the environment; (d) Promote plant growth in cadmium-polluted environments.

[0030] Preferably, its application method is as follows: The cadmium-resistant compound microbial agent was applied to cadmium-contaminated soil at a dosage of 210 g / L. 9 ~110 11 CFU / kg soil; The cadmium-resistant compound microbial agent is applied once before crop sowing. Crops include, but are not limited to, rice, wheat, corn, leafy vegetables, and alfalfa.

[0031] The specific implementation method is as follows: I. Cadmium resistance test of the strain 1. Prepare LB medium (1L) according to the following formula: 10g tryptone, 5g yeast extract, 10g sodium chloride, add distilled water to a final volume of 1000mL, adjust the pH to 7.0-7.2 with NaOH, and autoclave at 121℃ for 15 minutes. When preparing solid medium, add 15g agar.

[0032] 2. Add CdCl2·2.5H2O to sterilized LB medium to prepare Cd-containing solutions. 2+ LB medium containing cadmium at concentrations of 50 mg / L, 100 mg / L, 150 mg / L, and 200 mg / L.

[0033] 3. Inoculate Bacillus sp., Priestia megaterium, and Burkholderia cepacia into LB medium and cadmium-containing LB medium, respectively.

[0034] like Figure 1-3 As shown, although the growth of the three microorganisms in cadmium-containing LB medium was worse than that in LB medium, they could still grow, indicating that all three microorganisms had cadmium resistance.

[0035] II. The effect of compound microbial agents on the fixation of available cadmium in soil 1. A cadmium-contaminated soil sample was collected from a farmland, air-dried, and then sieved through a 2mm sieve. The total cadmium content was determined to be 3.2 mg / kg, the available cadmium content (DTPA extraction method) was 1.45 mg / kg, and the pH was 6.8.

[0036] 2. Configure the following processing groups: CK (control group): Add an equal volume of sterile water; T1 (single-strain group): Add Bacillus bacterial suspension (110) 8 CFU / g soil); T2 (single-strain group): Add *Priscilla megaterium* bacterial suspension (110) 8 CFU / g soil); T3 (single-strain group): Add Burkholderia bacterial suspension (110) 8 CFU / g soil); T4 (paired combination group): Bacillus + Priestella megaterium (1:1, total bacteria 110) 8 CFU / g soil); T5 (paired combination group): Bacillus + Burkholderia (1:1, total bacteria 110) 8 CFU / g soil); T6 (pairwise composite group): *Priscilla megaterium* + *Burkholderia burkholderia* (1:1, total bacteria 110). 8 CFU / g soil); T7 (Compound microbial agent group of this invention): Three kinds of bacteria are mixed in a 1:1:1 ratio (total bacteria 110). 8 CFU / g soil) Each group had three replicates. The bacterial solution was sprayed evenly onto the soil, mixed thoroughly, and the soil moisture content was adjusted to 60% of field capacity. The soil was then incubated at a constant temperature of 25°C. Samples were taken on day 28 to determine the available cadmium content in the soil.

[0037] 3. After 28 days of cultivation, the available cadmium content and reduction rate in the soil of each treatment group are shown in the table below: Processing group Available cadmium content (mg / kg) after 28 days Reduction rate of available cadmium (%) CK 1.42 2.07 T1 0.96 33.8 T2 0.91 37.2 T3 0.98 32.4 T4 0.72 50.3 T5 0.74 49.0 T6 0.70 51.7 T7 0.45 68.9 The results showed that the composite microbial agent (T7) of this invention had a significantly better effect on fixing available cadmium in the soil than the single-strain group and the pairwise composite group, with a reduction rate of available cadmium reaching 68.9%, demonstrating the synergistic effect among the three strains.

[0038] III. Effects of Compound Microbial Agents on Plant Growth in Cadmium-Contaminated Soils 1. Collect cadmium-contaminated soil from a farmland, with each container containing 2 kg of soil. Set up the following treatments: Control group (CK): Added an equal volume of sterile water; Compound microbial agent group: Liquid compound microbial agent was prepared according to the method of Example 4, and at 110 8 The dosage of CFU / g soil is applied.

[0039] Five replicates were used per group. Five alfalfa seeds (variety: alfalfa) were sown per pot after germination, and three seedlings were left per pot after emergence. The plants were managed as usual, and harvested after 30 days of growth. Plant height, fresh weight, dry weight, and cadmium content in the above-ground parts were measured.

[0040] 2. The results are as follows: Processing group Plant height (cm) Fresh weight (g / plant) Dry weight (g / plant) Cadmium content in aboveground parts (mg / kg dry weight) CK 22.52.0 12.81.5 2.450.25 2.850.21 Compound microbial agent 31.22.5 19.62.0 3.820.35 1.230.15 The results showed that after applying the compound microbial agent of the present invention, the plant height, fresh weight, and dry weight of alfalfa increased by 38.7%, 53.1%, and 55.9% respectively compared with the control group, while the cadmium content in the aboveground parts decreased by 56.8%. This indicates that the compound microbial agent of the present invention can not only effectively fix available cadmium in the soil and reduce the absorption of cadmium by plants, but also significantly promote plant growth.

[0041] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to any specific implementation. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A cadmium-resistant compound bacterial agent, characterized in that, This compound microbial agent comprises three microorganisms: Bacillus sp., Priestia megaterium, and Burkholderia cepacia. These three microorganisms do not exhibit antagonistic interactions and each can thrive in environments containing Cd. 2+ It grows in a suitable environment.

2. The cadmium-resistant compound microbial agent according to claim 1, characterized in that, In the compound microbial agent, the ratio of viable Bacillus, Priestella megaterium, and Burkholderia is (1-3):(0.5-2):(0.5-1.5).

3. The cadmium-resistant compound bacterial agent according to claim 2, characterized in that, The ratio of viable Bacillus, Priestella megaterium, and Burkholderia is 1:1:

1.

4. A method for preparing the cadmium-resistant compound microbial agent as described in any one of claims 1-3, characterized in that, Includes the following steps: S1, Bacillus, Priestella megaterium and Burkholderia were activated and cultured to obtain their respective seed solutions; S2, by mixing the seed liquids of Bacillus, Priestella megaterium and Burkholderia in a certain proportion, an anti-cadmium compound bacterial agent can be obtained.

5. The method for preparing an anti-cadmium compound bacterial agent according to claim 4, characterized in that, In step S1, the activation method is to inoculate Bacillus, Priestella megaterium and Burkholderia into LB medium and incubate them at 30°C for 24 hours.

6. The method for preparing an anti-cadmium compound bacterial agent according to claim 4, characterized in that, The method also includes the step of adding excipients. In step S2, the excipients are added simultaneously and mixed.

7. The method for preparing an anti-cadmium compound microbial agent according to claim 6, characterized in that, The excipients are one or more of the following: protective agents, carriers, dispersants, and nutrients.

8. The method for preparing an anti-cadmium compound bacterial agent according to claim 7, characterized in that, The protective agent is glycerol or trehalose; the carrier is diatomaceous earth or wheat bran; the dispersant is sodium carboxymethyl cellulose; and the nutrient is glucose or peptone.

9. The application of the anti-cadmium compound microbial agent as described in any one of claims 1-3, characterized in that, This compound microbial agent is used for at least one of the following purposes: (a) Remediation of cadmium-contaminated soil; (b) Reduce the content of available cadmium in the soil; (c) Fixing or transforming cadmium ions in the environment; (d) Promote plant growth in cadmium-polluted environments.

10. The application of the cadmium-resistant compound microbial agent according to claim 9, characterized in that, Its application method is as follows: The cadmium-resistant compound microbial agent was applied to cadmium-contaminated soil at a dosage of 210 g / L. 9 ~110 11 CFU / kg soil; The cadmium-resistant compound microbial agent is applied once before crop sowing, and the crops include, but are not limited to, rice, wheat, corn, leafy vegetables and alfalfa.

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