High-urease microbial gel, preparation method and method for repairing uranium-polluted soil by using high-urease microbial gel

Abstract

The invention discloses high-urease microbial gel, a preparation method and a method for repairing uranium-polluted soil by using the high-urease microbial gel, and belongs to the technical field of environmental pollution microbial treatment. The preparation method comprises the following steps: 1) mixing sodium alginate, urea and a water-retaining agent to obtain a carrier solution; 2) uniformly mixing the high urease bacteria solution in the logarithmic phase with the carrier solution according to a volume ratio of 5-25% to obtain a bacteria solution mixture; and (3) dropwise adding the bacterial liquid mixture into a cross-linking agent which is stirred at a constant speed for cross-linking and curing, and freeze-drying to obtain the high-urease microbial gel. The high-urease microbial gel is applied to the periphery of a root system of a uranium-enriched plant planted in uranium-contaminated soil to be repaired, and uranium-contaminated soil is repaired through combination of microorganisms and plants. Urease-producing bacteria in the high-urease microbial gel induce decomposition of urea to generate carbonate, uranyl ions in the soil are activated, the mobility of uranium in the soil is improved, and extraction of uranium in the soil by uranium-enriched plants is promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial treatment of environmental pollution, and particularly relates to a high-urease microbial gel, a preparation method thereof, and a method for repairing uranium-polluted soil. Background Art

[0002] Uranium is a toxic and radioactive heavy metal that poses a serious threat to the soil ecosystem. It not only pollutes the soil but may also affect the health of plants, animals, and humans through the cycling of the ecosystem. According to geochemical forms, uranium can be divided into two major categories: migratable and non-migratable. The non-migratable uranium in the soil mainly exists in the forms of iron and manganese oxide-bound, organic matter-bound, and mineral forms. Due to the difficulty of extraction, these non-migratable uranium are dispersed in the soil and difficult to recycle. In some uranium-polluted areas, the non-migratable uranium accounts for more than 90% of the total uranium concentration.

[0003] Currently, the treatment methods for uranium in the soil include physical-chemical, biological, and their combined methods. Physical-chemical remediation includes soil washing, electrokinetic remediation, and soil vitrification, etc., which remove uranium through chemical reactions or physical processes. Bioremediation utilizes the metabolic activities of microorganisms and plants to immobilize or transform uranium into low-toxic forms. Combined remediation combines multiple technologies, such as microorganism-plant combination, soil amendment-plant combination, and chelating agent-electrokinetic remediation combination, to overcome the limitations of single technologies and improve the remediation efficiency. However, factors such as the form of uranium, soil type, and environmental conditions will affect the remediation effect. The current remediation technologies still face challenges such as high cost and unstable effect. Especially when dealing with non-migratable uranium, its high proportion leads to poor direct enrichment and extraction effect.

[0004] Microbial gel is a technology that encapsulates or immobilizes microbial cells in microspherical carriers. Through physical or chemical methods, microbial cells are immobilized in tiny spherical carriers. These carriers are usually made of natural or synthetic polymer materials, such as sodium alginate, chitosan, polystyrene, etc. The microbial cells in these carriers can be bacteria, yeast, fungi, etc. Its advantages include stability (maintaining activity in harsh environments) and operability (simple preparation method and easy for large-scale production). Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies in the prior art and provide a high-urease microbial gel, a preparation method thereof, and a method for repairing uranium-polluted soil. The high-urease microbial gel prepared by the present invention can activate uranium in the soil through the carbonate provided by microorganisms, thereby assisting uranium-enriching plants to extract non-migratable uranium in the soil, realizing the removal of soil uranium pollution, and at the same time, uranium can be recovered after subsequent treatment of the harvested uranium-enriching plants.

[0006] The specific technical solutions adopted by the present invention are as follows:

[0007] In the first aspect, the present invention provides a method for preparing a high-urease microbial gel, and the steps are as follows:

[0008] S1: Mix sodium alginate, urea and a water-retaining agent to obtain a carrier solution; the mass fraction of urea in the carrier solution is 0.4% - 2%.

[0009] S2: Mix the high-urease bacterial liquid in the logarithmic phase with the carrier solution at a volume ratio of 5% - 25% to obtain a bacterial liquid mixture.

[0010] S3: Drop the bacterial liquid mixture into a cross-linking agent under uniform stirring for cross-linking and curing; freeze-dry the product to obtain a high-urease microbial gel; the cross-linking agent is a calcium chloride solution with a mass fraction of 1.5 - 2.5%; the mass ratio of sodium alginate to calcium chloride in step S1 is 0.1 - 0.4.

[0011] Preferably, the high-urease bacteria adopt Bacillus subtilis BL-416, which is preserved in the China Center for Type Culture Collection, with the preservation number of CCTCC FB 2024306, the preservation time of May 24, 2024, and the preservation address of the China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0012] Preferably, the water-retaining agent adopts polyglutamic acid, chitosan, carboxymethyl cellulose or polyacrylic acid; the mass fraction of the water-retaining agent is 1%.

[0013] Preferably, the stirring speed of the cross-linking agent in step S3 is 500 - 600 r / min.

[0014] Preferably, in the freeze-drying process of step S3, the cross-linked and cured product is first placed at -80°C for 2 h, and then freeze-dried in a freeze-dryer for 40 - 50 h.

[0015] In the second aspect, the present invention provides a high-urease microbial gel obtained by using the preparation method described in the first aspect.

[0016] In the third aspect, the present invention provides a method for activating uranium and enhancing phytoremediation of uranium-contaminated soil by using a high-urease microbial gel. Apply the high-urease microbial gel described in the second aspect around the roots of uranium-enriching plants planted in the uranium-contaminated soil to be repaired for microbial and plant combined remediation of uranium-contaminated soil; harvest the uranium-enriching plants after the end of the repair cycle; induce the decomposition of urea by urease-producing bacteria in the high-urease microbial gel to generate carbonate radicals, activate uranyl ions in the soil, improve the mobility of uranium in the soil, and promote the extraction of uranium in the soil by uranium-enriching plants.

[0017] Preferably, the high-urease microbial gel is applied within a range of 10 to 20 cm around the roots of uranium-enriching plants and is evenly dispersed.

[0018] Preferably, the application amount of the high-urease microbial gel is 7.5 to 15 g / m 2 .

[0019] Preferably, the uranium-enriching plants are Brassica juncea, Amaranthus retroflexus, Setaria viridis, Bidens pilosa, Polygonum hydropiper, Pteris multifida or Commelina communis.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention provides a preparation method of a high-urease microbial gel. By loading high-urease bacteria into microspheres, the mobility and bioavailability of heavy metal uranium in soil are significantly improved. Specifically, this method can convert immobile uranium in the forms of mineral state, organic matter-bound state, and iron and manganese oxide-bound state in soil into mobile uranyl carbonate (the mobile uranium in soil is increased to 25%-30%), so as to be applied as an auxiliary means for plant enrichment and remediation, and improve the absorption efficiency of plants for uranium.

[0022] (2) The high-urease microbial gel structure of the present invention has the characteristics of being loose, porous, moisture-absorbing and water-retaining, enabling the high-urease bacterial species to be slowly released into the soil and play its role of providing carbonate ions to migrate soil uranium in a long-term and stable manner. At the same time, during the urea decomposition process, the gel can also provide ammonia nitrogen for plants, further promoting plant growth, and can increase the extraction efficiency of uranium in soil by enriched plants by 45%-85%.

[0023] (3) The raw materials of the present invention are simple, the preparation method is simple, and the cost is low, effectively reducing the cost of soil uranium pollution remediation, meeting the large-scale remediation requirements of uranium-contaminated soil, and providing a practical solution for improving the state of uranium-contaminated soil. Description of the Drawings

[0024] Figure 1 Graph showing the change in the concentration of mobile uranium in soil affected by the high-urease microbial gel prepared in Example 1;

[0025] Figure 2 Graph comparing the uranium contents in the roots and above-ground parts of uranium-enriching plants after the treatments in Example 2 and Comparative Example 1;

[0026] Figure 3 Graph comparing the effects of different uranium concentrations on the growth of Bacillus subtilis in Example 3;

[0027] Figure 4 Graph comparing the effects of different uranium concentrations on the urea decomposition by Bacillus subtilis in Example 3;

[0028] Figure 5 It is a comparative diagram of the influence of Bacillus subtilis on the synthesis of carbonate under different concentrations of uranium in Example 3. Specific implementation manners

[0029] The present invention will be further elaborated and described below in conjunction with the accompanying drawings and specific implementation manners. The technical features of each implementation manner in the present invention can be combined correspondingly on the premise of no conflict with each other.

[0030] The high-urease bacteria used in the following examples is Bacillus subtilis BL-416. This strain is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC FB 2024306, the preservation time is May 24, 2024, and the preservation address is the China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0031] Example 1

[0032] In this example, a high-urease microbial gel was prepared, and the specific steps are as follows:

[0033] I. Inoculate 4 mL of the originally preserved Bacillus subtilis bacterial solution into 96 mL of bacterial growth medium, and culture it in a constant temperature shaker at 30 °C and 170 rpm for 8 h to obtain logarithmic-phase bacteria. Take 50 mL of the logarithmic-phase bacterial solution, centrifuge it at 3500 r / min for 5 min, pour off the upper-layer medium, add 50 mL of water and mix evenly to obtain the bacterial solution for preparing the high-urease microbial gel.

[0034] The bacterial growth medium includes 1 g / L peptone, 1 g / L glucose, 5 g / L sodium chloride, 2 g / L potassium dihydrogen phosphate and 10 g / L urea. Weigh peptone, glucose, sodium chloride, and potassium hydrogen phosphate according to the above formula, adjust the pH to 6.8 using NaOH and HCl solutions, heat and stir to completely dissolve the agar. Dispense it into 250 mL conical flasks, with each flask containing 150 mL of medium and a cotton plug, and perform high-pressure sterilization treatment (121 °C, 20 min, and urea is added when the medium cools to 60 °C).

[0035] II. Take 0.6 g of sodium alginate in 30 mL of ultrapure water, heat until completely dissolved, wait until it cools to 50 °C, add 0.6 g of urea, and add 0.5 g of polyglutamic acid to obtain a carrier solution.

[0036] III. After the solution in step II cools to 30 °C, mix 7.5 mL of the bacterial solution in step I with the carrier solution and make up the volume to 50 mL to obtain a bacterial solution mixture.

[0037] IV. Dissolve 2 g of calcium chloride in 100 mL of ultrapure water to obtain a crosslinking agent. Drop the bacterial liquid mixture into the crosslinking agent for crosslinking and curing. Stir the crosslinking agent using a magnetic stirrer at a stirring speed of 500 r / min. Slowly drop the bacterial liquid mixture using a syringe so that the bacterial liquid mixture dropped into the crosslinking agent is spherical. Wash the obtained microspheres three times with sterile water and filter the water thoroughly, then place them in a -80°C refrigerator and freeze for 2 h, and then lyophilize for 40 - 50 h to obtain high-urease microbial gel.

[0038] Place the high-urease biogel prepared in this example in uranium-contaminated soil, keep the soil moisture content at 60% of the soil saturated moisture content, and continue for 15 days. Take samples every 3 days to detect the concentration of mobile uranium in the soil. The uranium concentration of the uranium-contaminated soil used in the experiment is 153 mg / kg. Figure 1 It is a graph showing the change in the concentration of mobile uranium in the soil affected by the high-urease microbial gel obtained in this example.

[0039] According to Figure 1 As can be seen, after adding the high-urease microbial gel to the uranium-contaminated soil, the concentration of mobile uranium in the soil reaches 36.41 mg / kg on the third day, and the activation rate is 23.80%. By the 9th day, the concentration of mobile uranium reaches the highest value of 40.85 mg / kg, and the activation rate increases to 26.70% at this time and then tends to be stable. The experiment shows that the activation effect of the high-urease microbial gel on uranium in the soil can be maintained for at least 15 days.

[0040] Example 2

[0041] In this example, the high-urease microbial gel prepared in Example 1 is used for an experiment on the combined extraction of uranium from soil with plants, and the specific steps are as follows:

[0042] Adopt an indoor pot experiment. Select the uranium-enriched plant as Brassica juncea L. Put the sieved uranium-contaminated soil (≤2 mm) into each plastic pot. The uranium concentration of the uranium-contaminated soil used in the experiment is 153 mg / kg.

[0043] Plant 10 evenly sized Brassica juncea L. seeds in each pot, adjust the soil water content to 50% of the field water holding capacity, and place a petri dish under each pot to collect the possible leachate during the phytoremediation process. After Brassica juncea L. has grown for 50 days, apply the high-urease microbial gel to a position 10 - 20 cm around the roots of Brassica juncea L., and the application amount is 10 g / m 2 . 14 days after applying the high-urease microbial gel, cut the branches of the plants above the soil surface, wash them with deionized water, and then separate and wash the roots from the soil until all soil particles are removed. Freeze-dry the plants and weigh them to measure the biomass, and detect the uranium content in the plants.

[0044] Comparative Example 1

[0045] In this comparative example, an experiment on the combined use of Bacillus subtilis bacterial solution and plants to extract uranium from soil was carried out as follows:

[0046] An indoor pot experiment was adopted. The uranium-enriched plant selected was Brassica juncea L. Sifted uranium-contaminated soil (≤2 mm) was placed in each plastic pot. The uranium concentration of the uranium-contaminated soil used in the experiment was 153 mg / kg

[0047] 10 evenly sized Brassica juncea L. seeds were planted in each pot. The soil water content was adjusted to 50% of the field water holding capacity, and a petri dish was placed under each pot to collect the possible leachate during the phytoremediation process. 50 days after the Brassica juncea L. was planted and grew, the bacterial solution of Bacillus subtilis in the logarithmic phase was applied to a position 10 - 20 cm around the root system of Brassica juncea L. 14 days after the application of the bacterial solution, the branches of the plant were cut above the soil surface, rinsed with deionized water, and then the roots were separated and rinsed from the soil until all soil particles were removed. The plants were freeze-dried and weighed to measure the biomass, and the uranium content in the plants was detected.

[0048] The experimental results in Example 2 and Comparative Example 1 are as Figure 2 shown, where the blank group only planted Brassica juncea L. According to Figure 2 it can be seen that for the uranium concentration in the roots, the uranium concentration in the roots of Brassica juncea L. planted in the blank group was 428.28 mg / kg, about 2.80 times the soil uranium concentration. The uranium concentration in the roots of Brassica juncea L. planted in the group with the application of Bacillus subtilis bacterial solution in Comparative Example 1 was 628.09 mg / kg, about 4.11 times the soil uranium concentration. The uranium concentration in the roots of Brassica juncea L. planted with the application of high-urease microbial gel in Example 2 was 781.92 mg / kg, about 5.11 times the soil uranium concentration. For the uranium concentration in the above-ground parts, the uranium concentration in the above-ground parts of Brassica juncea L. in the blank group was 96.82 mg / kg, about 0.63 times the soil uranium concentration. The uranium concentration in the above-ground parts of Brassica juncea L. in Comparative Example 1 was 183.20 mg / kg, about 1.20 times the soil uranium concentration. The uranium concentration in the above-ground parts of Brassica juncea L. in Example 2 was 205.29 mg / kg, about 1.34 times the soil uranium concentration.

[0049] Root uranium concentration comparison: The root uranium concentration in Example 2 was the highest, reaching 781.92 mg / kg, which was 1.88 times that of the blank group and 1.24 times that of Comparative Example 1. The root uranium concentration in Comparative Example 1 was 628.09 mg / kg, which was 1.55 times that of the blank group. This indicates that the high-urease microbial gel significantly enhances the uranium enrichment ability of Indian mustard roots. Shoot uranium concentration comparison: The shoot uranium concentration in Example 2 was 205.29 mg / kg, which was 1.34 times that of the blank group and 1.12 times that of Experimental Group 1. The shoot uranium concentration in Comparative Example 1 was 183.20 mg / kg, which was 1.20 times that of the blank group. This indicates that both the high-urease microbial gel and the bacterial solution can improve the uranium enrichment ability of the shoots of Indian mustard, but the effect of the high-urease microbial gel is more significant.

[0050] Comprehensive analysis shows that the uranium concentrations in the roots and shoots of Example 2 were significantly higher than those of the other two groups, indicating that the high-urease microbial gel can effectively improve the bioavailability of uranium in the soil, thereby enhancing the uranium enrichment ability of Indian mustard. Compared with Comparative Example 1 that only added Bacillus subtilis bacterial solution, the uranium extraction amount of the group applying the high-urease microbial gel in Example 2 was higher, indicating that the gel was superior to the bacterial solution in terms of stability and effect. The uranium concentration of the blank group was relatively low, indicating that the uranium enrichment ability of Indian mustard itself was limited, but by adding the high-urease microbial gel or bacterial solution, its enrichment ability could be significantly enhanced.

[0051] Example 3

[0052] This example verifies the effect of different uranium concentrations on this Bacillus subtilis as follows:

[0053] I. Prepare a uranyl nitrate solution with a concentration of 3000 mg / L, and dissolve 45 mg of uranyl nitrate in 15 mL of ultrapure water.

[0054] II. Prepare 5 conical flasks, add 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of the uranyl nitrate solution respectively, then add bacterial growth medium to 96 mL, and inoculate 4 mL of bacterial suspension to obtain bacterial culture solutions with uranyl concentrations of 30 mg / L, 60 mg / L, 90 mg / L, 120 mg / L, and 150 mg / L.

[0055] III. Shake at 130 rpm at room temperature for 6 days, and measure OD 600 , carbonate concentration, bicarbonate concentration, and urea concentration every 24 hours. The results are as Figure 3 , Figure 4 and Figure 5 shown.

[0056] According to Figure 3It can be seen that under the conditions of each concentration gradient of uranyl nitrate, the activity of the bacteria did not show a significant downward trend. This finding indicates that even in an environment with a high concentration of uranyl nitrate, the bacteria can still maintain relatively stable physiological activity, and the internal metabolic process is not significantly inhibited. According to Figure 4 and Figure 5 it can be seen that under the conditions of different concentrations of uranyl nitrate, the urea decomposition efficiency of the bacteria also did not show obvious differences. This shows that the high concentration of uranyl nitrate did not interfere with the urea decomposition metabolic pathway of the bacteria, and the bacteria can still efficiently decompose urea into products such as carbonate radicals.

[0057] The above-described embodiments are only a preferred solution of the present invention, but they are not intended to limit the present invention. Those of ordinary skill in the relevant technical field can also make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all technical solutions obtained by adopting equivalent replacement or equivalent transformation methods fall within the protection scope of the present invention.

Claims

1. A preparation method of a high-urease microbial gel, characterized in that, The steps are as follows: S1: Mix sodium alginate, urea, and a water retention agent to obtain a carrier solution; the mass fraction of urea in the carrier solution is 0.4% - 2%; S2: Mix the high-urease bacteria liquid in the logarithmic phase with the carrier solution at a volume ratio of 5% - 25% to obtain a bacteria liquid mixture; S3: Drop the bacteria liquid mixture into a cross-linking agent under uniform stirring for cross-linking and curing; freeze-dry the product to obtain a high-urease microbial gel; the cross-linking agent is a calcium chloride solution with a mass fraction of 1.5 - 2.5%; the mass ratio of sodium alginate to calcium chloride in step S1 is 0.1 - 0.

4.

2. The preparation method of the high-urease microbial gel according to claim 1, characterized in that, The high-urease bacteria used is Bacillus subtilis BL-416, which is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC FB 2024306, the preservation time is May 24, 2024, and the preservation address is the China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

3. The preparation method of the high-urease microbial gel according to claim 1, characterized in that, The water retention agent used is polyglutamic acid, chitosan, carboxymethyl cellulose, or polyacrylic acid; the mass fraction of the water retention agent is 1%.

4. The preparation method of the high-urease microbial gel according to claim 1, characterized in that, In step S3, the stirring speed of the cross-linking agent is 500 - 600 r / min.

5. The preparation method of the high-urease microbial gel according to claim 1, characterized in that, In step S3, during the freeze-drying process, first place the cross-linked and cured product at -80°C for 2 h, and then freeze-dry it in a freeze-dryer for 40 - 50 h.

6. A high-urease microbial gel obtained by using the preparation method according to any one of claims 1 - 5.

7. A method for activating uranium by using high-urease microorganism gel and enhancing phytoremediation of uranium pollution, characterized in that, Apply the high-urease microbial gel according to claim 6 around the roots of uranium-enriched plants planted in the uranium-contaminated soil to carry out the combined remediation of uranium-contaminated soil by microorganisms and plants; harvest the uranium-enriched plants after the end of the remediation cycle; induce the decomposition of urea by urease-producing bacteria in the high-urease microbial gel to produce carbonate ions, activate the uranyl ions in the soil, improve the mobility of uranium in the soil, and promote the extraction of uranium in the soil by uranium-enriched plants.

8. The method for activating uranium by high-urease microorganism gel and enhancing phytoremediation of uranium pollution according to claim 7, characterized in that, The high-urease microbial gel is applied within a range of 10 - 20 cm around the roots of uranium-enriched plants and is evenly dispersed.

9. The method for activating uranium by high-urease microorganism gel and enhancing phytoremediation of uranium pollution according to claim 7, characterized in that, The application amount of the high-urease microorganism gel is 7.5 to 15 g / m 2 .

10. The method for activating uranium by high-urease microorganism gel and enhancing phytoremediation of uranium pollution according to claim 7, characterized in that, The uranium-enriched plants are Brassica juncea, Amaranthus retroflexus, Setaria viridis, Bidens pilosa, Polygonum hydropiper, Pteris multifida, or Commelina communis.

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