A microbial agent based on arsenic-converting microorganisms for long-term oxidation and stabilization of soil arsenic pollution

Abstract

The present invention belongs to the technical field of soil pollution remediation, and specifically relates to a bacterial agent based on arsenic conversion microorganisms for long-term oxidation and stabilization of soil arsenic pollution. The bacterial agent based on arsenic conversion microorganisms for long-term oxidation and stabilization of soil arsenic pollution includes a modified guar gum carrier and a bacterial solution, and the preparation of the modified guar gum carrier includes the following steps: taking guar gum, phosphorus tribromide, and pyridine in toluene at 5-20°C for 10-12h, and treating to obtain brominated guar gum; taking brominated guar gum and N,N,N',N'-tetramethyl-1,4-butanediamine in tetrahydrofuran for 16-24h, and treating to obtain a modified guar gum carrier. The modified guar gum and the bacterial solution act together to increase the bacterial solution repair speed, and can adsorb and aggregate arsenic ions in the soil, so that the bacterial solution can quickly oxidize trivalent arsenic ions into a pentavalent arsenic ion state with low toxicity and low mobility.

Description

Technical Field

[0001] The present invention belongs to the technical field of soil pollution remediation, and specifically relates to a bacterial agent based on arsenic conversion microorganisms for long-term oxidation and stabilization of soil arsenic pollution. Background Art

[0002] The current situation of arsenic contamination in soil is severe, posing a huge threat to human health and the ecological environment. Arsenic is a toxic semi-metallic element. Long-term exposure to arsenic-contaminated soil can lead to a variety of health problems, such as skin diseases, neurological diseases and even cancer. At present, with the increase of industrialization and agricultural activities, the problem of arsenic contamination in soil is becoming increasingly serious, especially in some areas with rich mineral resources or intensive industrial activities, where soil arsenic concentrations exceeding safety limits are common.

[0003] According to the tightness of arsenic binding in the soil, arsenic in the soil can be divided into different binding forms, mainly including water-soluble, exchangeable, bound (such as iron-bound, calcium-bound, aluminum-bound) and residual arsenic. It is generally believed that water-soluble and exchangeable arsenic has strong bioavailability and is easily absorbed by plants, while residual arsenic has poor bioavailability and is not easily absorbed by plants. However, when the physical and chemical conditions of the soil change or the phase or particle size of arsenic-bound minerals changes under the action of soil microorganisms, the arsenic in the soil can be released into effective arsenic. Changes in the binding forms of arsenic in the soil and its distribution ratio often lead to changes in plant absorption of arsenic and changes in the bioavailability of arsenic in the soil.

[0004] At present, there are two main ways to remediate arsenic-contaminated soil: one is to change the existing form of arsenic in the contaminated soil, transforming it from an active state to a stable state, thereby reducing the mobility and bioavailability of arsenic in the environment and weakening its toxicity to plants and animals; the other is to remove arsenic from the contaminated soil to achieve the dual purpose of recycling and reducing heavy metals in the soil, so that its remaining concentration is close to or reaches the background value. At present, the remediation and treatment technology of arsenic-contaminated soil at home and abroad is developing rapidly, mainly including the following: engineering measures, including soil import method, soil replacement method and soil turning method; physical remediation technology, including solidification / stabilization, physical separation remediation, vitrification remediation, electric remediation, etc.; chemical remediation technology, including in situ / ex situ chemical leaching technology, soil performance improvement technology, etc.; biological remediation technology, including animal remediation, microbial remediation, plant remediation, etc.

[0005] Among them, in terms of engineering measures, soil remediation is fast and effective, and can quickly reduce pollution concentrations, but it is costly, requires a lot of excavation and transportation, may damage the soil structure, and is not suitable for large-scale pollution; physical remediation has a stable effect, no risk of secondary pollution, and can be used for deep soil remediation, but it is costly, technically complex, and has a certain impact on soil properties; chemical remediation is fast and effective, and can target specific pollutants, but it may cause secondary pollution, has a high cost, and has potential impacts on soil ecology; biological remediation is low-cost, environmentally friendly, and conducive to the recovery of soil ecosystems, but the remediation speed is slow, the effect is greatly affected by environmental factors, the remediation effect is unstable, and the scope of application is limited.

[0006] Compared with other remediation technologies, microbial remediation technology has significant advantages in the field of environmental remediation, but it also has some limitations. In order to overcome these limitations, it is necessary to further strengthen the research and development of microbial remediation technology, improve the degradation ability and adaptability of microorganisms, improve the evaluation and monitoring methods of remediation effects, and explore more efficient and economical remediation methods. Summary of the invention

[0007] One of the purposes of the present invention is to provide a bacterial agent based on arsenic-converting microorganisms for long-term oxidation and stabilization of soil arsenic pollution, which can quickly and effectively oxidize trivalent arsenic in the soil into pentavalent arsenic, thereby increasing the speed of bioremediation.

[0008] To achieve one of the purposes of the present invention, the technical solution adopted by the present invention is:

[0009] A microbial agent for long-term oxidation and stabilization of soil arsenic pollution based on arsenic conversion microorganisms, the microbial agent comprising a modified guar gum carrier and a bacterial solution, wherein the structure of the modified guar gum carrier is:

[0010] .

[0011] Furthermore, the preparation process of the modified guar gum carrier comprises the following steps:

[0012] ;

[0013] (1) guar gum, phosphorus tribromide and pyridine are reacted in toluene at 5-20°C for 10-12h to obtain brominated guar gum;

[0014] (2) Brominated guar gum and N,N,N',N'-tetramethyl-1,4-butanediamine are reacted in tetrahydrofuran for 16 to 24 hours to obtain a modified guar gum carrier.

[0015] Furthermore, the mass ratio of the guar gum to phosphorus tribromide and pyridine is 2:1-1.5:0.88-1.

[0016] Furthermore, the mass ratio of the brominated guar gum to N,N,N',N'-tetramethyl-1,4-butanediamine is 2:0.5-0.55.

[0017] Furthermore, the preparation of the bacterial solution includes the following steps:

[0018] 1) Strain screening: The strain is Pseudomonas taiwanensis (Pseudomonas taiwanensis, purchased from China Industrial Microbiological Culture Collection Center);

[0019] 2) Preparation of culture medium: the culture medium is one of nutrient broth agar and tryptone soy broth, sterilized in a steam pressure autoclave at 0.1 MPa and 121° C. for 20 min;

[0020] 3) Strain activation: Take out the Taiwan Pseudomonas strains stored at -80~-70°C, slowly thaw them in an ice bath, inoculate the thawed strains into 90~100 times the volume of the culture medium, place it in a constant temperature shaking incubator at 160~180rpm and 30~32°C for 24~48h to obtain an activated bacterial solution;

[0021] 4) Expanding the culture and collecting the bacterial liquid: inoculating the activated bacterial liquid into a fermenter filled with 20 times the volume of culture medium of the activated bacterial liquid, and culturing for 24 hours at 30° C., 180 r / min and aeration volume of 1 vvm to obtain the bacterial liquid.

[0022] The second object of the present invention is to provide a method for preparing a bacterial agent for long-term oxidation and stabilization of arsenic pollution in soil based on arsenic-converting microorganisms.

[0023] To achieve one of the purposes of the present invention, the technical solution adopted by the present invention is:

[0024] The preparation method of the above-mentioned bacterial agent based on arsenic-converting microorganisms for long-term oxidation and stabilization of soil arsenic pollution comprises the following steps: then mixing the mixed bacterial solution and the carrier in a mass ratio of 2:3, stirring at 180-200 rpm for 3-5 minutes, and then drying at 15-20°C, with a moisture content of 6-10wt% after drying, and packaging to obtain the bacterial agent based on arsenic-converting microorganisms for long-term oxidation and stabilization of soil arsenic pollution.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. A bacterial agent based on arsenic-converting microorganisms for long-term oxidation and stabilization of arsenic pollution in soil, comprising a modified guar gum carrier and a bacterial solution. The preparation principle of the modified guar gum is as follows: a portion of the alcohol hydroxyl groups on the modified guar gum are subjected to a halogenation reaction to obtain brominated guar gum, and the brominated guar gum is further reacted with a tertiary amine to prepare a quaternary ammonium salt to obtain the modified guar gum.

[0027] 2. The modified guar gum and the bacterial solution work together to improve the repair speed of the bacterial solution: The modified guar gum has quaternary ammonium cations, which can improve the electrostatic interaction and adsorption performance of inorganic arsenite ions. It has high adsorption efficiency, large adsorption capacity, and is green and environmentally friendly. After the modified guar gum and the bacterial solution are combined, the arsenite ions in the soil can be adsorbed and aggregated, so that the bacterial solution can quickly oxidize trivalent arsenic ions into low-toxic, low-mobility pentavalent arsenic ions. DETAILED DESCRIPTION

[0028] The technical scheme of the present invention is further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0029] Example 1

[0030] A microbial agent for long-term oxidation and stabilization of soil arsenic pollution based on arsenic conversion microorganisms comprises a modified guar gum carrier and a bacterial solution:

[0031] The preparation process of the modified guar gum carrier comprises the following steps:

[0032] ;

[0033] (1) 2 g of guar gum was dispersed in a reaction bottle containing 11 mL of toluene, and 1.2 g of phosphorus tribromide was added dropwise to the reaction bottle at 5° C. After the addition was complete, 920 mg of pyridine was added to the reaction bottle, and the reaction solution was gradually heated from 5° C. to 20° C. and reacted for 10 h; the reaction was quenched with 5 wt % sodium hydroxide solution until the pH of the reaction solution was 7, and then filtered. After filtering, the reaction solution was washed with deionized water and ethanol in turn to obtain brominated guar gum;

[0034] (2) Take 2 g of brominated guar gum and 520 mg of N,N,N',N'-tetramethyl-1,4-butanediamine in 11 mL of tetrahydrofuran and react for 18 h. Filter the solvent and wash with deionized water and ethanol in turn to obtain modified guar gum.

[0035] The preparation of the bacterial solution comprises the following steps:

[0036] 1) Strain screening: The strain is Pseudomonas taiwanensis (Pseudomonas taiwanensis, purchased from China Industrial Microbiological Culture Collection Center);

[0037] 2) Preparation of culture medium: The culture medium is nutrient broth agar, which is sterilized in a steam pressure sterilizer at 0.1 MPa and 121°C for 20 min;

[0038] 3) Strain activation: Take out the Taiwan Pseudomonas strains stored at -80°C and slowly thaw them in an ice bath; inoculate the thawed strains into 100 times the volume of culture medium, place in a constant temperature shaking incubator and shake and culture at 170 rpm and 31°C for 36 hours to obtain an activated bacterial solution;

[0039] 4) Expanding the culture and collecting the bacterial solution: inoculating the activated bacterial solution into a fermenter filled with 20 times the volume of nutrient broth agar medium of the activated bacterial solution, and culturing for 24 hours at 30° C., 180 r / min and aeration volume of 1 vvm to obtain the bacterial solution.

[0040] The preparation process of the bacterial agent based on arsenic-converting microorganisms for long-term oxidation and stabilization of arsenic pollution in soil includes the following steps: mixing the bacterial solution and the modified guar gum carrier in a mass ratio of 2:3, stirring at 190 rpm for 4 minutes, and then drying at 18°C, with a moisture content of 8wt% after drying, and packaging to obtain the bacterial agent based on arsenic-converting microorganisms for long-term oxidation and stabilization of arsenic pollution in soil.

[0041] Example 2

[0042] A microbial agent for long-term oxidation and stabilization of soil arsenic pollution based on arsenic conversion microorganisms comprises a modified guar gum carrier and a bacterial solution:

[0043] The preparation process of the modified guar gum carrier comprises the following steps:

[0044] (1) 2 g of guar gum was dispersed in a reaction bottle containing 10 mL of toluene, and 1 g of phosphorus tribromide was added dropwise to the reaction bottle at 5° C. After the addition was complete, 880 mg of pyridine was added to the reaction bottle, and the reaction solution was gradually heated from 5° C. to 20° C. and reacted for 12 h; the reaction was quenched with 5 wt % sodium hydroxide solution until the pH of the reaction solution was 7, and then filtered. After filtering, the reaction solution was washed with deionized water and ethanol in turn to obtain brominated guar gum;

[0045] (2) Take 2 g of brominated guar gum and 500 mg of N,N,N',N'-tetramethyl-1,4-butanediamine in 10 mL of tetrahydrofuran and react for 24 h. Filter the solvent and wash with deionized water and ethanol in turn to obtain modified guar gum.

[0046] The preparation of the bacterial solution comprises the following steps:

[0047] 1) Strain screening: The strain is Pseudomonas taiwanensis (Pseudomonas taiwanensis, purchased from China Industrial Microbiological Culture Collection Center);

[0048] 2) Culture medium preparation: the culture medium is tryptone soy broth, which is sterilized in a steam pressure autoclave at 0.1 MPa and 121°C for 20 min;

[0049] 3) Strain activation: Take out the Taiwan Pseudomonas strains stored at -70°C, slowly thaw them in an ice bath, inoculate the thawed strains into 100 times the volume of culture medium, place in a constant temperature shaking incubator at 160 rpm and 30°C for 48 hours, and obtain an activated bacterial solution;

[0050] 4) Expanding the culture and collecting the bacterial solution: inoculating the activated bacterial solution into a fermenter filled with 20 times the volume of tryptone soy broth medium of the activated bacterial solution, culturing for 24 hours at 30° C., 180 r / min and aeration volume of 1 vvm to obtain the bacterial solution.

[0051] The preparation process of the bacterial agent based on arsenic-converting microorganisms for long-term oxidation and stabilization of arsenic pollution in soil includes the following steps: mixing the bacterial solution and the carrier in a mass ratio of 2:3, stirring at 180 rpm for 3 minutes, and then drying at 15°C, with a moisture content of 6wt% after drying, and packaging to obtain the bacterial agent based on arsenic-converting microorganisms for long-term oxidation and stabilization of arsenic pollution in soil.

[0052] Example 3

[0053] A microbial agent for long-term oxidation and stabilization of soil arsenic pollution based on arsenic conversion microorganisms comprises a modified guar gum carrier and a bacterial solution:

[0054] The preparation process of the modified guar gum carrier comprises the following steps:

[0055] (1) 2 g of guar gum was dispersed in a reaction bottle containing 12 mL of toluene, and 1.5 g of phosphorus tribromide was added dropwise to the reaction bottle at 5° C. After the addition was complete, 1 g of pyridine was added to the reaction bottle, and the reaction solution was gradually heated from 5° C. to 20° C. and reacted for 11 h; the reaction was quenched with 5 wt % sodium hydroxide solution until the pH of the reaction solution was 7, and then filtered. After filtering, the reaction solution was washed with deionized water and ethanol in turn to obtain brominated guar gum;

[0056] (2) Take 2 g of brominated guar gum and 550 mg of N,N,N',N'-tetramethyl-1,4-butanediamine in 12 mL of tetrahydrofuran and react for 24 h. Filter the solvent and wash with deionized water and ethanol in turn to obtain modified guar gum.

[0057] The preparation of the bacterial solution comprises the following steps:

[0058] 1) Strain screening: The strain is Pseudomonas taiwanensis (Pseudomonas taiwanensis, purchased from China Industrial Microbiological Culture Collection Management Center);

[0059] 2) Preparation of culture medium: The culture medium is nutrient broth agar, which is sterilized in a steam pressure sterilizer at 0.1 MPa and 121°C for 20 min;

[0060] 3) Strain activation: Take out the Taiwan Pseudomonas strains stored at -70°C, slowly thaw them in an ice bath, inoculate the thawed strains into 90 times the volume of culture medium, place in a constant temperature shaking incubator at 180 rpm and 32°C for 24 hours, and obtain an activated bacterial solution;

[0061] 4) Expanding the culture and collecting the bacterial solution: inoculating the activated bacterial solution into a fermenter filled with 20 times the volume of nutrient broth agar medium of the activated bacterial solution, and culturing for 24 hours at 30° C., 180 r / min and aeration volume of 1 vvm to obtain the bacterial solution.

[0062] The preparation process of the bacterial agent based on arsenic-converting microorganisms for long-term oxidation and stabilization of arsenic pollution in soil includes the following steps: mixing the bacterial solution and the carrier in a mass ratio of 2:3, stirring at 200 rpm for 5 minutes, and then drying at 20°C, with a moisture content of 10wt% after drying, and packaging to obtain the bacterial agent based on arsenic-converting microorganisms for long-term oxidation and stabilization of arsenic pollution in soil.

[0063] Test Example 1

[0064] Experiment on the removal of arsenic in soil by the bacterial agent of the present invention:

[0065] Soil pretreatment: Take 5 flower pots, each with 2 kg of high-concentration arsenic-contaminated soil, numbered A1-A5, detect the initial arsenic concentration and record it. The specific detection method is: weigh 2 g of soil sample into a 100 mL conical flask, add 600 μL of deionized water to moisten the soil, let it stand for 10 minutes, then add 5 mL of 65 wt% concentrated nitric acid, 3 mL of 30 wt% sulfuric acid, and 1 mL of 70 wt% perchloric acid in sequence, stir at room temperature for 3 hours, and then heat to boiling until the mixed solution becomes grayish white and then cool; then add 5 mL of 65 wt% concentrated nitric acid and 10 mL of deionized water, heat to boiling until the solution evaporates to 5 mL; when the conical flask is cooled to 30°C, add 15 mL of deionized water to fully dissolve the soil, and then filter it with filter paper; then add deionized water to wash the soil, filter it with neutral filter paper, and then make the volume up to 50 mL with deionized water, and finally use a total reflection X-ray fluorescence spectrometer to test the arsenic content in the liquid.

[0066] Sampling steps: Take 2g of soil sample into a 50mL beaker, add 15mL of deionized water, soak the soil for 5min, stir under a magnetic stirrer for 2h, let it stand for 1h, filter through 3 layers of gauze, collect the leachate, determine the arsenic content in the leachate, and calculate the arsenic stabilization rate of the leachate: arsenic stabilization rate (%) = 1-[arsenic content / 1d arsenic content].

[0067] Example 1-3 Process of treating soil with bacterial agent: 0.2 g and 0.1 g of ferrous sulfate prepared in Example 1-3 were added to 1 kg of arsenic-contaminated soil sample, respectively, stirred evenly, and cultured for 90 days to ensure that the soil moisture content was about 20-30%;

[0068] Process of treating soil with bacterial solution: add the bacterial solution equivalent to 0.2g of the bacterial agent prepared in Example 1 and 0.1g of ferrous sulfate to 1kg of arsenic-contaminated soil sample, stir evenly, and culture for 90 days to ensure that the soil moisture content is about 20-30%;

[0069] Guar gum inoculant (the difference from the method of preparing the inoculant in Example 1 is that the guar gum inoculant uses guar gum equal to the modified guar gum in Example 1) to treat soil process: 0.2 g of guar gum inoculant and 0.1 g of ferrous sulfate are added to 1 kg of arsenic-contaminated soil sample, stirred evenly, and cultured for 90 days to ensure that the soil moisture content is about 20-30%.

[0070] The eluted arsenic content (mg / mL) and arsenic stabilization rate (%) of A1-A5 were detected after 1d, 15d, 30d, 60d, and 90d, as shown in Table 1;

[0071] Table 1 Effect of the bacterial agent of the present invention on arsenic content in soil

[0072]

[0073] As shown in Table 1, compared with the bacterial solution group and the guar gum bacterial agent group, the bacterial agent group of Examples 1-3 maintained a relatively superior arsenic stabilization ability in 1-90 days, indicating that the present invention has achieved a relatively obvious performance in the long-term oxidation of bacterial agents. The bacterial solution group has been acting on the arsenic content in the soil in 1-90 days, but its effect range is relatively limited, and its arsenic stabilization rate after 90 days is only 40.28, which is far lower than 65.78%~67.04% of Examples 1-3; the arsenic stabilization effect of the guar gum bacterial agent in 1-90 days is slightly better than that of the bacterial solution group, but there is a relatively obvious gap with the Example 1-3 group. This shows that the present invention has a relatively outstanding arsenic stabilization ability.

[0074] The above are only preferred embodiments of the present invention, and are not limited to the above examples. For those skilled in the art, various changes and variations are possible under the principle of the present invention. Any modifications and improvements made should be considered within the scope of protection of the present invention.

Claims

1. A microbial agent for long-term oxidation and stabilization of soil arsenic pollution based on arsenic-converting microorganisms, characterized in that: The bacterial agent includes a modified guar gum carrier and a bacterial solution, wherein the bacterial solution contains Pseudomonas Taiwanensis, and the structure of the modified guar gum carrier is: 。 2. The microbial agent for long-term oxidation and stabilization of soil arsenic pollution based on arsenic-converting microorganisms according to claim 1 is characterized in that: The preparation process of the modified guar gum carrier comprises the following steps: ； (1) guar gum, phosphorus tribromide and pyridine are reacted in toluene at 5-20°C for 10-12h to obtain brominated guar gum; (2) Brominated guar gum and N,N,N',N'-tetramethyl-1,4-butanediamine are reacted in tetrahydrofuran for 16 to 24 hours, and a modified guar gum carrier is obtained after treatment.

3. The microbial agent for long-term oxidation and stabilization of soil arsenic pollution based on arsenic-converting microorganisms according to claim 2 is characterized in that: The mass ratio of the guar gum to phosphorus tribromide and pyridine is 2:1-1.5:0.88-1.

4. The microbial agent for long-term oxidation and stabilization of soil arsenic pollution based on arsenic-converting microorganisms according to claim 2, characterized in that: The mass ratio of the brominated guar gum to N,N,N',N'-tetramethyl-1,4-butanediamine is 2:0.5-0.

55.

5. The microbial agent for long-term oxidation and stabilization of soil arsenic pollution based on arsenic-converting microorganisms according to claim 1, characterized in that: The preparation of the bacterial solution comprises the following steps: 1) Strain screening: The strain is Pseudomonas Taiwanensis; 2) Preparation of culture medium: The culture medium is one of nutrient broth agar and tryptone soy broth, which is sterilized in a steam pressure autoclave at 0.1 MPa and 121° C. for 20 min; 3) Strain activation: Take out the Taiwan Pseudomonas strains stored at -80~-70°C, slowly thaw them in an ice bath, inoculate the thawed strains into 90~100 times the volume of the culture medium, place it in a constant temperature shaking incubator at 160~180rpm and 30~32°C for 24~48h to obtain an activated bacterial solution; 4) Expanding the culture and collecting the bacterial liquid: inoculating the activated bacterial liquid into a fermenter filled with 20 times the volume of culture medium of the activated bacterial liquid, and culturing for 24 hours at 30° C., 180 r / min and aeration volume of 1 vvm to obtain the bacterial liquid.

6. The method for preparing a bacterial agent for long-term oxidation and stabilization of soil arsenic pollution based on arsenic-converting microorganisms according to any one of claims 1 to 5 comprises the following steps: The mixed bacterial solution and the carrier are then mixed in a mass ratio of 2:3, stirred at 180-200 rpm for 3-5 minutes, and then dried at 15-20° C., with a moisture content of 6-10 wt % after drying, and packaged to obtain a bacterial agent based on arsenic-converting microorganisms for long-term oxidation and stabilization of soil arsenic pollution.

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