A method for mitigating the risk of environmental arsenic pollution

Abstract

The present application relates to the technical field of soil remediation, and discloses a risk reduction method for environmental arsenic pollution, which comprises the following steps: preparing a culture medium, culturing a nitrogen-fixing microbial flora in an antibiotic-resistant manner, co-culturing the nitrogen-fixing microbial flora and arsenic-polluted soil, and analyzing the influence of soil arsenic conversion efficiency, so that the arsenic content in the soil is significantly reduced, the arsenic resistance of the nitrogen-fixing microbial flora reaches 500 mg / kg, the harm of soil arsenate is reduced, the safe management and control of arsenic-polluted soil can be realized, the nitrogen-fixing microbial flora can significantly reduce the content of trivalent arsenic in the soil, the trivalent arsenic with strong toxicity in the soil is more converted into pentavalent arsenic with weak toxicity, the conversion efficiency can reach more than 94.36%, and the risk reduction method for environmental arsenic pollution is successfully constructed, which has strong functionality and good popularization.

Description

Technical Field

[0001] This invention belongs to the field of soil remediation, specifically relating to a method for mitigating the risk of environmental arsenic pollution. Background Technology

[0002] Arsenic (As) is a toxic metal found in groundwater, surface water, sediments, and soil. It exists in more than 200 minerals in the Earth's crust, mainly in the form of arsenates, with a small portion existing as arsenites, arsenides, oxides, and elemental arsenic. The elemental form can indicate the toxicity and bioavailability of the element. In soil, arsenic exists in different forms, including free arsenic, solid form (precipitate), adsorbed form, and exchanged form. Arsenic in soil mainly exists in inorganic forms of As(III) and As(V) and a small amount of organic forms. The toxicity of arsenic mainly depends on its valence state and chemical composition.

[0003] The valence state and chemical composition of arsenic are the main factors affecting arsenic toxicity. Inorganic arsenic varies significantly in terms of chemical properties and biological toxicity. Arsenite (As(III)), one of the most toxic arsenic compounds, commonly known as arsenic trioxide or arsenic sulfide, has a significant effect on mitochondrial respiration. When the human body ingests food containing arsenic, it is mainly distributed in the liver, nails and other parts through blood circulation and binds to proteins, inhibiting the related activities of proteases and DNA repair. This leads to cellular metabolic disorders, nutritional deficiencies, and causes the greatest harm to the nervous system. The most serious acute arsenic poisoning may cause symptoms such as fever, loss of appetite, hepatomegaly, and arrhythmia, and may even lead to death. Chronic exposure to arsenic poisoning may cause damage to the peripheral nervous system, manifested as loss of sensation, paralysis, muscle spasms and other symptoms, and may even affect the blood system.

[0004] Arsenic pollution in soil poses a serious threat to agricultural production and human health. Arsenic is a toxic heavy metal element that can enter the soil through natural processes or human activities (such as mining and industrial emissions). Long-term accumulation can lead to a decline in soil quality, hinder crop growth, and even cause poisoning incidents. Therefore, it is crucial to improve arsenic-contaminated soil. Currently, physical, chemical, and biological methods are commonly used to remediate arsenic-contaminated soil. However, physical (chemical) methods such as changing farming practices, leaching soil, and adding antagonists and amendments still result in secondary pollution. After implementation, these methods are found to be labor-intensive, time-consuming, and difficult to fundamentally eliminate the harm caused by arsenic.

[0005] This application proposes a method for mitigating the risk of environmental arsenic pollution, thereby improving upon the aforementioned deficiencies. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for reducing the risk of environmental arsenic pollution that can fundamentally eliminate the harm caused by arsenic.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A method for mitigating the risk of environmental arsenic pollution includes culture medium preparation, arsenic-tolerant culture of nitrogen-fixing microbial communities, and co-culture of nitrogen-fixing microbial communities with arsenic-contaminated soil. The method for mitigating the risk of environmental arsenic pollution of this invention includes the following steps:

[0009] S1. Culture medium preparation: LB medium was selected as the solid and liquid culture medium. 10g tryptone, 5g yeast extract and 5g sodium chloride were weighed and diluted to 1L with ultrapure water. 15g agar was added to solidify the mixture. The mixture was then dispensed into Erlenmeyer flasks and sealed with sealing film.

[0010] As a preferred method, the sealed conical flask is placed in an autoclave and sterilized at 121°C and 100 kPa for 20 minutes to obtain liquid and solid culture media, which can then be cooled for later use.

[0011] S2. Antibiotic resistance culture of nitrogen-fixing microorganisms: Nitrogen-fixing bacteria were placed in LB liquid medium and cultured in a constant temperature shaking incubator at 25℃ and 160 rpm. If the medium became turbid after 12 hours, the nitrogen-fixing bacteria had grown. 1 ml of the above nitrogen-fixing bacterial suspension was placed in mediums containing 10 mg / L oxytetracycline and 10 mg / L sulfadiazine and cultured. If the medium became turbid after 12 hours, the nitrogen-fixing bacteria had grown. The antibiotic concentration in the medium was gradually increased, using antibiotic concentration gradients of 50 mg / L, 100 mg / L, 200 mg / L, 500 mg / L, and 1000 mg / L to train the nitrogen-fixing bacteria to resist antibiotics. Finally, nitrogen-fixing bacteria that could grow under conditions of 1000 mg / L oxytetracycline and 1000 mg / L sulfadiazine were obtained as the experimental strains.

[0012] Preferably, the nitrogen-fixing microbial community mainly includes rhizobia and free-living nitrogen-fixing bacteria.

[0013] S3. Co-culture of nitrogen-fixing microbial communities with arsenic-contaminated soil: Selected nitrogen-fixing microbial strains resistant to 1000 mg / L oxytetracycline and 1000 mg / L sulfadiazine, as well as uncultured resistant nitrogen-fixing strains, were added to soils with exogenously added trivalent arsenic at concentrations of 100 mg / kg, 300 mg / kg, and 500 mg / kg for co-culture for up to 35 days. Sterile culture medium was used as a control. On the 35th day of culture, samples were taken to determine the existing forms and concentrations of arsenic.

[0014] According to the above-mentioned technical solution, the present invention provides a method for reducing the risk of environmental arsenic pollution, which has the following beneficial effects:

[0015] (1) This invention provides a method for mitigating the risk of environmental arsenic pollution. By preparing culture media, cultivating nitrogen-fixing microbial communities to antibiotic resistance, and co-culturing nitrogen-fixing microbial communities with arsenic-contaminated soil, combined with the analysis of the impact on soil arsenic conversion efficiency, the arsenic content in the soil is significantly reduced. Furthermore, the arsenic resistance of nitrogen-fixing microbial communities can reach 500 mg / kg, thereby reducing the harm of soil arsenate and enabling safe management of arsenic-contaminated soil.

[0016] (2) This invention provides a risk reduction method for environmental arsenic pollution. By using nitrogen-fixing microbial communities, the content of trivalent arsenic in the soil can be significantly reduced, which promotes the conversion of more of the more toxic trivalent arsenic in the soil into less toxic pentavalent arsenic. The conversion efficiency can reach more than 94.36%. The invention has successfully constructed a risk reduction method for environmental arsenic pollution, which is highly functional and has good applicability. Attached Figure Description

[0017] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0018] Figure 1 This is a schematic diagram illustrating the transformation of a method for mitigating the risk of environmental arsenic pollution in an embodiment of this application;

[0019] Figure 2 This is a schematic diagram showing the change in arsenic content after 35 days of cultivation in an embodiment of this application. Detailed Implementation

[0020] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0021] A method for mitigating the risk of environmental arsenic pollution is proposed. The nitrogen-fixing microbial community mainly includes rhizobia and free-living nitrogen-fixing bacteria. Rhizobia can convert nitrogen in the air into forms such as ammonia or nitrogenous acid that can be absorbed by plants for their use. Free-living nitrogen-fixing bacteria convert nitrogen in the air into nitrogen compounds that can be used by plants through nitrogen fixation. They can improve soil texture, promote the decomposition and recycling of soil organic matter, improve soil fertility, and maintain the stability of the ecosystem. In agricultural production and soil management, it is important to protect and promote the function of nitrogen-fixing microorganisms in order to achieve sustainable agricultural development and ecological environmental protection.

[0022] Example 1: Please refer to Figures 1-2 The specific embodiments of the present invention are as follows:

[0023] A method for mitigating the risk of environmental arsenic pollution, comprising the following steps:

[0024] S1. Culture medium preparation: LB medium was selected as the solid and liquid culture medium. 10g tryptone, 5g yeast extract and 5g sodium chloride were weighed and diluted to 1L with ultrapure water. The solid culture medium was prepared in the same way as above, but 15g agar was added to solidify it. The medium was dispensed into Erlenmeyer flasks, sealed with sealing film, and placed in an autoclave. It was sterilized at 121℃ and 100KPa for 20min to obtain liquid and solid culture media. The media were cooled and ready for use.

[0025] S2. Antibiotic-resistant culture of nitrogen-fixing microbial community: Nitrogen-fixing bacteria were placed in LB liquid medium and cultured in a constant temperature shaking incubator at 25℃ and 160r / min. If the medium became turbid after 12h, the nitrogen-fixing bacteria had grown.

[0026] Then, 1 ml of the above-mentioned nitrogen-fixing bacteria culture was placed in a medium containing 10 mg / L oxytetracycline and 10 mg / L sulfadiazine and cultured. If the medium became turbid after 12 hours, the nitrogen-fixing bacteria had grown. The antibiotic concentration in the medium was then gradually increased to train the nitrogen-fixing bacteria to resist antibiotics by culturing at antibiotic concentration gradients of 50 mg / L, 100 mg / L, 200 mg / L, 500 mg / L, and 1000 mg / L. Finally, nitrogen-fixing bacteria that could grow under conditions of 1000 mg / L oxytetracycline and 1000 mg / L sulfadiazine were obtained as the experimental strain.

[0027] S3. Co-culture of nitrogen-fixing microbial communities with arsenic-contaminated soil: Selected nitrogen-fixing microbial strains resistant to 1000 mg / L oxytetracycline and 1000 mg / L sulfadiazine, as well as uncultured resistant nitrogen-fixing strains, were added to soil with exogenously added trivalent arsenic at a concentration of 100 mg / kg for co-culture for up to 35 days. Sterile culture medium was used as a control. On the 35th day of culture, samples were taken to test the existing forms and concentrations of arsenic.

[0028] Example 2: Please refer to Figures 1-2 The specific embodiments of the present invention are as follows:

[0029] A method for mitigating the risk of environmental arsenic pollution, comprising the following steps:

[0030] M1. Culture medium preparation: LB medium was selected as the solid and liquid culture medium. 10g tryptone, 5g yeast extract and 5g sodium chloride were weighed and diluted to 1L with ultrapure water. The solid culture medium was prepared in the same way as above, but 15g agar was added to solidify it. The medium was dispensed into Erlenmeyer flasks, sealed with sealing film, and placed in an autoclave. It was sterilized at 121℃ and 100KPa for 20min to obtain liquid and solid culture media. The media were cooled and used for later use.

[0031] M2. Antibiotic-resistant culture of nitrogen-fixing microbial community: Nitrogen-fixing bacteria were placed in LB liquid medium and cultured in a constant temperature shaking incubator at 25°C and 160 r / min. If the medium became turbid after 12 h, the nitrogen-fixing bacteria had grown.

[0032] Then, 1 ml of the above-mentioned nitrogen-fixing bacteria culture was placed in a medium containing 10 mg / L oxytetracycline and 10 mg / L sulfadiazine and cultured. If the medium became turbid after 12 hours, the nitrogen-fixing bacteria had grown. The antibiotic concentration in the medium was then gradually increased to train the nitrogen-fixing bacteria to resist antibiotics by culturing at antibiotic concentration gradients of 50 mg / L, 100 mg / L, 200 mg / L, 500 mg / L, and 1000 mg / L. Finally, nitrogen-fixing bacteria that could grow under conditions of 1000 mg / L oxytetracycline and 1000 mg / L sulfadiazine were obtained as the experimental strain.

[0033] M3. Co-culture of nitrogen-fixing microbial communities with arsenic-contaminated soil: Selected nitrogen-fixing microbial strains resistant to 1000 mg / L oxytetracycline and 1000 mg / L sulfadiazine, as well as uncultured resistant nitrogen-fixing strains, were added to soil with exogenously added trivalent arsenic at a concentration of 300 mg / kg for co-culture for up to 35 days. The sterile culture medium treatment served as a control. On the 35th day of culture, samples were taken to test the existing forms and concentrations of arsenic.

[0034] Example 3: Please refer to Figures 1-2 The specific embodiments of the present invention are as follows:

[0035] A method for mitigating the risk of environmental arsenic pollution, comprising the following steps:

[0036] K1. Culture medium preparation: LB medium was selected as the solid and liquid culture medium. 10g tryptone, 5g yeast extract and 5g sodium chloride were weighed and diluted to 1L with ultrapure water. The solid culture medium was prepared in the same way as above, but 15g agar was added to solidify it. The mixture was dispensed into Erlenmeyer flasks, sealed with sealing film, and placed in an autoclave. It was sterilized at 121℃ and 100KPa for 20min to obtain liquid and solid culture media. The media were cooled and used for later use.

[0037] K2. Antibiotic-resistant culture of nitrogen-fixing microbial community: Nitrogen-fixing bacteria were placed in LB liquid medium and cultured in a constant temperature shaking incubator at 25℃ and 160r / min. If the medium became turbid after 12h, the nitrogen-fixing bacteria had grown.

[0038] Then, 1 ml of the above-mentioned nitrogen-fixing bacteria culture was placed in a medium containing 10 mg / L oxytetracycline and 10 mg / L sulfadiazine and cultured. If the medium became turbid after 12 hours, the nitrogen-fixing bacteria had grown. The antibiotic concentration in the medium was then gradually increased to train the nitrogen-fixing bacteria to resist antibiotics by culturing at antibiotic concentration gradients of 50 mg / L, 100 mg / L, 200 mg / L, 500 mg / L, and 1000 mg / L. Finally, nitrogen-fixing bacteria that could grow under conditions of 1000 mg / L oxytetracycline and 1000 mg / L sulfadiazine were obtained as the experimental strain.

[0039] K3. Co-culture of nitrogen-fixing microbial communities with arsenic-contaminated soil: Selected nitrogen-fixing microbial strains resistant to 1000 mg / L oxytetracycline and 1000 mg / L sulfadiazine, as well as uncultured resistant nitrogen-fixing strains, were added to soil with exogenously added trivalent arsenic at a concentration of 500 mg / kg for co-culture for up to 35 days. The sterile culture medium treatment served as a control. On the 35th day of culture, samples were taken to test the existing forms and concentrations of arsenic.

[0040] In Examples 1 to 3, nitrogen-fixing microorganisms were co-cultured with arsenic-contaminated soils of different concentration gradients (100 mg / kg, 300 mg / kg, and 500 mg / kg) for 35 days. The tolerance characteristics of these microorganisms to arsenic were observed, and soil samples collected after 35 days of culture were used to determine the concentration and speciation of arsenic. Through the implementation of Examples 1 to 3 and statistical analysis, the transformation of exogenous trivalent arsenic in soil by nitrogen-fixing microorganisms was as follows: Figure 1 and Figure 2 As shown, the experimental results indicate that the screened arsenic-resistant nitrogen-fixing microorganisms can promote the conversion of highly toxic trivalent arsenic into less toxic pentavalent arsenic.

[0041] This invention solves the problem of soil arsenic pollution. By preparing culture media, cultivating antibiotic-resistant nitrogen-fixing microbial communities, and co-culturing nitrogen-fixing microbial communities with arsenic-contaminated soil, combined with the analysis of the impact on soil arsenic conversion efficiency, this invention significantly reduces the arsenic content in the soil and enables the nitrogen-fixing microbial communities to achieve arsenic tolerance of 500 mg / kg, thereby reducing the harm of soil arsenate and enabling safe management of arsenic-contaminated soil.

[0042] In the description of this invention, it should be understood that the indicated orientation or positional relationship is based on the orientation or positional relationship shown in the accompanying drawings, and is only for the convenience of describing this invention and simplifying the description, and is not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0043] The above are merely specific embodiments of this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A method for reducing the risk of environmental arsenic pollution, comprising medium preparation, arsenic-tolerant culture of a nitrogen-fixing microbial community, and co-culture of the nitrogen-fixing microbial community with arsenic-contaminated soil, the method comprising the following steps: S1. Medium preparation, selecting LB medium as a solid or liquid medium, weighing tryptone, yeast extract, and sodium chloride, and then adding ultrapure water to a final volume of 1 L, followed by adding 15 g of agar to solidify the medium, and then dispensing the medium into conical flasks, sealing the flasks with a sealing film, and placing the sealed flasks in an autoclave to obtain the liquid and solid media, which are then cooled and stored for use; S2. Arsenic-tolerant culture of a nitrogen-fixing microbial community, placing the nitrogen-fixing bacteria in LB liquid medium and culturing in a constant-temperature shaking incubator, and after 12 hours, if the medium is turbid, the nitrogen-fixing bacteria are growing; or taking 1 ml of the above-mentioned nitrogen-fixing bacteria solution and placing it in a medium containing oxytetracycline or a medium containing sulfadiazine for culture, and after 12 hours, if the medium is turbid, the nitrogen-fixing bacteria are growing, and the concentration of antibiotics in the medium is gradually increased, and finally, the nitrogen-fixing bacteria that can grow in the presence of 1000 mg / L oxytetracycline and 1000 mg / L sulfadiazine are obtained as the experimental bacteria; S3. Co-culture of a nitrogen-fixing microbial community with arsenic-contaminated soil, adding the better nitrogen-fixing microbial strains selected from the strains that can tolerate 1000 mg / L oxytetracycline and 1000 mg / L sulfadiazine and uncultured resistant nitrogen-fixing strains to soil that has been exogenously supplemented with trivalent arsenic, with sterile medium as a control; The weighed tryptone, yeast extract, and sodium chloride are 10 g tryptone, 5 g yeast extract, and 5 g sodium chloride, and the volume is made up to 1 L with ultrapure water, followed by the addition of 15 g of agar to solidify the medium; The nitrogen-fixing microbial community mainly includes rhizobium and free-living nitrogen-fixing bacteria. The antibiotic concentration is used to train the resistance of nitrogen-fixing bacteria to antibiotics at a gradient of 50 mg / L, 100 mg / L, 200 mg / L, 500 mg / L, and 1000 mg / L.

2. A method of risk reduction of environmental arsenic contamination according to claim 1, characterized in that: The concentration of the soil that has been exogenously supplemented with trivalent arsenic is 100 mg / kg, 300 mg / kg, or 500 mg / kg.

3. The method of risk mitigation of environmental arsenic contamination according to claim 1, characterized in that: The nitrogen-fixing microbial strains and uncultured resistant nitrogen-fixing strains are added to soil that has been exogenously supplemented with trivalent arsenic and co-cultured for 35 days, and samples are taken on the 35th day of culture to determine the forms and concentrations of arsenic.

4. The method of claim 1, wherein the method is used to reduce the risk of environmental arsenic contamination. ​

Images