Method for Remediating Lead-Contaminated Saline Soil by Biomineralization of Pseudomonas stutzeri

Abstract

The present invention discloses a method for using Pseudomonas stutzeri for biomineralization to repair lead-contaminated saline-alkali soil, belonging to the technical field of soil heavy metal remediation. In the present invention, Pseudomonas stutzeri, calcium acetate and calcium citrate that have passed the activity test and adaptability test are applied to the lead-contaminated saline-alkali soil, and the biomineralization of Pseudomonas stutzeri is utilized to repair the lead-contaminated soil. During the process of Pseudomonas stutzeri biosynthesizing calcium carbonate precipitate using calcium acetate and calcium citrate, lead ions in the soil can be precipitated in the form of coprecipitation or locked in the crystal lattice of calcium carbonate, thereby effectively reducing the mobility and toxicity of soil lead and achieving the purpose of in-situ passivation of soil lead and safe utilization of soil. All the medicaments involved in the present invention can be completely degraded or precipitated, and will not exacerbate the salinization of the soil. The present invention is applicable to the remediation of saline-alkali soil contaminated with lead at medium and low concentrations.

Description

Technical Field

[0001] The invention relates to a method for repairing lead pollution in salinized soil by utilizing Pseudomonas stutzeri biomineralization, and belongs to the technical field of soil heavy metal repair. Background Art

[0002] Salinized soil is widely distributed in my country, from tropical to cold zones, from coastal to inland, from humid areas to extremely arid desert areas. Northwest China, North China, Northeast China and the eastern coastal areas are the main concentrated distribution areas of salinized soil in my country. The total area of salinized soil in my country is about 3600×10 4 hm 2 , accounting for 4.88% of the country's available land area. Many salinized soils are contaminated by heavy metals (such as lead). The salt in salinized soils, including NaCl, Na2SO4, MgCl2, MgSO4, etc., will activate soil heavy metals and increase the ecological risk of heavy metals.

[0003] At present, a variety of soil lead pollution remediation methods have been developed, including physical remediation, chemical remediation and biological remediation. The physical remediation methods mainly include soil import method and soil replacement method. This type of engineering construction requires a large amount of work and the cost of pollution remediation is high. In addition, soil replacement can easily destroy the soil structure of the local environment, causing a decrease in soil fertility. It is only suitable for remediating soils with a small area and high lead pollution concentration. It is not economical for regional, large-area and low-concentration lead-contaminated soils. The chemical remediation method mainly adds passivators to the contaminated soil to enhance the soil's own adsorption and precipitation, thereby reducing the bioavailability of lead in the soil and reducing the migration of pollutants. It can change the problem of soil lead pollution to a certain extent, but chemical passivators have problems such as destroying the physical and chemical properties of the soil and poor durability of the remediation effect. Microbial remediation is a low-cost and environmentally friendly method. Microbial remediation methods for repairing soil lead pollution include biosorption and biomineralization. Biosorption has a good effect on the adsorption and passivation of lead, but other cations existing in large quantities in the environment will compete for adsorption sites, and the adsorbed lead will be re-released after cell apoptosis or dispersion, making it difficult to truly apply to lead-contaminated sites. Biomineralization has the characteristics of high remediation efficiency, low cost, and simple operation.

[0004] Traditional bio-mineralization technology uses urea-type microorganisms to induce carbonate mineralization technology, but the large-scale use of urea will lead to soil ammonia nitrogen problems. A large amount of ammonium ions will form metal-ammine complexes with heavy metal cations such as Pb, which will in turn aggravate the dissolution of heavy metals and cause soil compaction problems. In addition, traditional bio-mineralization technology uses calcium chloride as a calcium source, which will leave a large amount of chloride ions after soil remediation, aggravating soil salinization, and is not suitable for heavy metal remediation of salinized soils.

[0005] In summary, in the face of saline-alkali soil contaminated by lead with a large area but medium and low concentrations, there is an urgent need for an economical, efficient, and stable remediation method. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for remediating lead pollution in saline-alkali soil by using the biomineralization process of Pseudomonas stutzeri in view of the deficiencies of the prior art. This method uses Pseudomonas stutzeri to degrade calcium acetate and calcium citrate, and in-situ mineralizes to repair soil heavy metal lead pollution. Compared with traditional biomineralization, it will not cause nitrogen pollution problems and will not exacerbate soil salinization problems.

[0007] In order to achieve the above-mentioned invention purpose, the specific technical solutions used in the present invention are as follows:

[0008] The present invention provides a method for remediating lead pollution in saline-alkali soil by using Pseudomonas stutzeri biomineralization, which is characterized in that Pseudomonas stutzeri (Pseudomonas stutzeri) that has passed the activity test and adaptability test is used as a remediation agent, and calcium acetate and calcium citrate are used as calcium sources. The remediation agent, calcium acetate, calcium citrate, urea, and potassium dihydrogen phosphate are dissolved in water to form a soil remediation solution; the soil remediation solution is applied to the soil to be remediated, and the soil is kept in an aerobic environment; Pseudomonas stutzeri degrades the calcium source to biosynthesize calcium carbonate precipitate, and during the biosynthesis process, lead ions in the soil are precipitated or locked in the calcium carbonate lattice in the form of coprecipitation, thereby reducing the mobility and toxicity of lead in the soil and achieving the purpose of in-situ remediation of lead pollution in saline-alkali soil by biomineralization.

[0009] Preferably, the above-mentioned Pseudomonas stutzeri is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC K B 20081858.

[0010] Preferably, the above-mentioned soil remediation solution contains 0.01-0.05 g / L of Pseudomonas stutzeri powder, 1-5 g / L of calcium acetate, 1-5 g / L of calcium citrate, 0.4-2 g / L of urea, and 0.05-0.25 g / L of potassium dihydrogen phosphate.

[0011] Preferably, the specific methods of the above-mentioned activity test and adaptability test are as follows:

[0012] Disperse Pseudomonas stutzeri into physiological saline to obtain an initial bacterial solution; inoculate the initial bacterial solution into a first culture medium for growth activity culture; detect the OD600 value of the bacterial solution, and take the first bacterial solution with an OD600 value greater than 1.5 to complete the growth activity detection;

[0013] Inoculate the bacterial liquid that has completed the growth activity detection into the second culture medium for constant temperature culture; when the OD600 value of the bacterial liquid in the second culture medium is greater than 1.5, take a small amount of the precipitate, wash it, and then add a hydrochloric acid solution with a concentration of 1 M for adaptability test; if bubbles appear in the precipitate, the adaptability test is passed.

[0014] The first culture medium includes 2.5 g / L calcium acetate, 2.5 g / L calcium citrate, 1.0 g / L urea, 0.2 g / L potassium dihydrogen phosphate, and 5.0 g / L sodium chloride.

[0015] The preparation method of the second culture medium is as follows: Add 2.5 g / L calcium acetate, 2.5 g / L calcium citrate, 1.0 g / L urea, and 0.2 g / L potassium dihydrogen phosphate to the soil solution, and then filter it with a 0.45 μm filter membrane.

[0016] Further, the mass concentration of Pseudomonas stutzeri in the above initial bacterial liquid is 2 g / L; the inoculation amount of the initial bacterial liquid in the first culture medium is 1%; the inoculation amount of the bacterial liquid in the second culture medium is 1%.

[0017] Further, the preparation method of the above soil solution is as follows: Disperse the soil to be repaired that has passed through a 100-mesh sieve into water at a mass concentration of 100 g / L to obtain a soil mixture; oscillate the soil mixture at 30 °C and 170 rpm for 8 hours, and then filter it with a 0.45 μm filter membrane to obtain the supernatant, which is the soil solution.

[0018] Preferably, the EC value of the soluble salt concentration in the above soil to be repaired is less than 50 dS / m, the soil pH range is 5 - 8, the soil lead pollution depth is less than 40 cm, and the soil lead concentration is less than 100 mg / kg.

[0019] Preferably, the application amount of the above soil repair liquid is 1 - 4 L per square meter of soil.

[0020] Preferably, the above soil to be repaired is plowed with a rotary tiller, and the soil repair liquid is sprayed while plowing; the number of times of plowing and spraying the soil repair liquid is 2 - 3 times.

[0021] Preferably, the temperature maintained during the repair process of the above soil to be repaired is 10 - 35 °C, the soil moisture content is 10 - 30%, and the repair period is 5 - 10 days.

[0022] Compared with the prior art, the advantages of the present invention are as follows:

[0023] (1) Compared with the traditional biogenic mineralization technology that uses calcium chloride as the calcium source, which leads to a large amount of chloride ions remaining in the soil after soil remediation, thus exacerbating the soil salinization problem, calcium acetate and calcium citrate are used as the calcium sources in the present invention, and Pseudomonas stutzeri is utilized to degrade calcium acetate and calcium citrate. All the medicaments in the soil remediation solution provided by the present invention can be completely degraded or precipitated, without salt residue, and will not exacerbate the soil salinization.

[0024] (2) Urea and potassium dihydrogen phosphate in the soil remediation solution of the present invention not only provide nutrient elements for the growth and metabolism of Pseudomonas stutzeri, but also can be used as nitrogen fertilizer and phosphate fertilizer to promote the growth of plants in the soil. In addition, some low-molecular organic substances secreted by the growth of microorganisms are also beneficial to plant growth. Therefore, the present invention can not only repair the lead pollution of saline-alkali soil, but also promote the restoration of soil vegetation or increase the yield of crops.

[0025] (3) When the present invention is applied to sulfate-type saline-alkali soil, calcium ions in the soil remediation solution are not only an important raw material for the biogenic mineralization of Pseudomonas stutzeri, but also can combine with sulfate ions in the soil to form calcium sulfate precipitate (gypsum), thereby reducing the soil salinity, being beneficial to increasing soil fertility and promoting plant growth.

[0026] (4) The present invention utilizes the biogenic mineralization of Pseudomonas stutzeri to repair the lead pollution of saline-alkali soil. Compared with physical and chemical methods, it has the advantages of simple process, convenient operation, low cost, quick effect, no secondary pollution, and no damage to the soil structure, and is particularly suitable for the remediation of saline-alkali soil contaminated by lead with medium and low concentrations. Detailed implementation mode

[0027] The following specific examples are used to further explain and illustrate the present invention, but the protection scope of the present invention is not limited thereto.

[0028] The inventive concept of the present invention is to inoculate the Pseudomonas stutzeri bacterial solution in the logarithmic growth phase into the saline-alkali lead-contaminated soil. In the loose soil with sufficient air and nutrients, Pseudomonas stutzeri can grow rapidly and quickly degrade calcium acetate and calcium citrate to synthesize calcium carbonate precipitate. During the biosynthesis process of calcium carbonate precipitate, lead ions in the soil can be precipitated in the form of coprecipitation or locked in the crystal lattice of calcium carbonate, thereby effectively reducing the mobility and toxicity of soil lead and achieving the purpose of in-situ passivation of soil lead and safe utilization of soil. The following specific examples are used to demonstrate the technical effects that the present invention can achieve.

[0029] The Pseudomonas stutzeri strain used in the following embodiments of the present invention is deposited at the China Center for Type Culture Collection, with the deposit address being Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit date is October 25, 2005, and the deposit number is CCTCC KB 20081858. The remaining reagents in the following embodiments can all be commercial products of analytical purity or above available on the market.

[0030] Example

[0031] In this example, a small-scale soil test was conducted to verify the effect of Pseudomonas stutzeri in biogenic mineralization for the remediation of lead-contaminated saline soil, as follows:

[0032] (1) Obtaining the lead-contaminated saline soil to be remediated, the steps are as follows:

[0033] Collect the surface soil (0 - 20 cm) of a saline agricultural land in North China, pass it through a 100-mesh sieve to remove impurities such as plastics, gravel, and plant roots. The soil moisture content is 15.4%, the soil pH is 7.66, the salinity is 32.1 dS / m, and the concentrations of sulfate, bicarbonate, and chloride ions are 8.15 mg / kg, 0.13 mg / kg, and 0.11 mg / kg respectively. Take 40 g of the above soil, add 20 mL of lead chloride solution (lead concentration is 100 mg / L), and stir evenly with a glass rod. Dry it in a ventilated and cool place to obtain the soil to be remediated with a lead contamination concentration of 50 mg / kg.

[0034] (2) Activity test of Pseudomonas stutzeri, including growth activity detection and mineralization activity detection, the steps are as follows:

[0035] Weigh 0.1 g of Pseudomonas stutzeri bacterial powder and disperse it in 50 mL of normal saline (0.9% sodium chloride solution) to prepare 50 mL of the initial bacterial solution. Pipette 1 mL of the initial bacterial solution and add it to 100 mL of the culture medium. The culture medium includes 2.5 g / L calcium acetate, 2.5 g / L calcium citrate, 1.0 g / L urea, 0.2 g / L potassium dihydrogen phosphate, and 5.0 g / L sodium chloride. Then place the culture medium containing the bacterial solution in a constant temperature shaker and shake it at 30 °C and 170 rpm for 24 hours. After the culture is completed, monitor the OD 600 value. If the OD 600 value is greater than 1.5, then the growth activity detection is passed; otherwise, it indicates that the growth activity of the bacterial agent is insufficient or it has been contaminated. Finally, take a small amount of the cultured precipitate, wash it 3 times with water, and add 1 mL of hydrochloric acid solution with a concentration of 1 M. If a large number of bubbles are generated in the precipitate, then the mineralization activity (i.e., the activity of biosynthesizing calcium carbonate) detection is passed; otherwise, it indicates that the mineralization activity of the bacterial agent is insufficient or it has been contaminated.

[0036] The process of preparing the culture medium in the above steps is as follows: First, dissolve 0.1 g of urea, 0.02 g of potassium dihydrogen phosphate, and 0.5 g of sodium chloride in 50 mL of deionized water, and sterilize it at high temperature for 20 minutes at 121 °C. Dissolve 0.25 g of calcium acetate and 0.25 g of calcium citrate in 50 mL of deionized water, and filter it through a 0.45 μm filter membrane to remove the miscellaneous bacteria in the solution. After the high-temperature sterilized solution is cooled to room temperature, in a laminar flow hood, mix the above two 50 mL solutions to prepare 100 mL of culture medium.

[0037] (3) The adaptability test of Pseudomonas stutzeri is carried out as follows:

[0038] Pass the soil to be repaired obtained in step (1) through a 100-mesh sieve, weigh 10 g of the sieved soil, disperse it in 100 mL of deionized water, and oscillate it for 8 hours at 30 °C and 170 rpm to obtain a soil solution. Filter the above soil solution through a 0.45 μm filter membrane to obtain a sterile soil solution.

[0039] Using the above soil solution, configure the culture medium as follows: Take 100 mL of the sterile soil solution, and sequentially add 0.25 g of calcium acetate, 0.25 g of calcium citrate, 0.1 g of urea, and 0.02 g of potassium dihydrogen phosphate; filter the above solution through a 0.45 μm filter membrane again to obtain a sterile culture medium prepared from the soil solution.

[0040] Take 100 mL of the sterile culture medium prepared from the soil solution, inoculate 1 mL of the initial bacterial solution, place it in a constant temperature shaker, and oscillate and culture it for 24 hours at 30 °C and 170 rpm. After the culture is completed, monitor the OD 600 value. Finally, take a small amount of the cultured precipitate, wash it 3 times with water, drop 1 mL of hydrochloric acid solution (concentration 1 M), and observe the generation of bubbles in the precipitate. If the OD 600 value is greater than 1.5, and a large number of bubbles are generated after adding hydrochloric acid to the precipitate, then the adaptability test of the soil environment is passed, otherwise it means that the used microbial agent is not suitable for the soil to be repaired.

[0041] (4) Preparation of the soil remediation solution, the steps are as follows:

[0042] Use the Pseudomonas stutzeri that has passed the activity test and adaptability test as the remediation microbial agent, weigh 0.05 g of Pseudomonas stutzeri powder, and disperse it in 20 mL of normal saline (0.9% sodium chloride solution). Dissolve 0.25 g of calcium acetate, 0.25 g of calcium citrate, 0.1 g of urea, and 0.02 g of potassium dihydrogen phosphate in deionized water, and then add 1 mL of the normal saline containing the microbial agent, and mix well to prepare the soil remediation solution.

[0043] (5) Soil remediation, the steps are as follows:

[0044] Take the soil to be repaired obtained in step (1), divide it into two equal parts, each part being 20 g. One group is used as the experimental group, and the other group is used as the control group. In the experimental group, 5 mL of the soil repair solution obtained in step (4) is uniformly added and thoroughly mixed with the soil to be repaired; in the control group, 5 mL of deionized water is uniformly added and also thoroughly mixed with the soil. After mixing, the soil is evenly spread in a glass petri dish, and the soil thickness is less than 5 mm. Place the above soil in an incubator at 25°C. The two glass petri dishes are placed side by side with their mouths open and repaired for 7 days.

[0045] (6) The results of soil repair are as follows:

[0046] Using the Tessier five-step extraction method, the repaired soil is continuously chemically extracted, and the contents of various forms of lead (Pb) are analyzed. The results show that after 7 days of repair, the exchangeable Pb in the soil of the experimental group decreased to 0.86 mg / kg (the passivation rate was 98.3%), and that in the control group only decreased to 17.9 mg / kg (the passivation rate was only 64.2%). Compared with the control group, the stable Pb forms such as carbonate-bound, iron and manganese oxide-bound, organic-bound, and residual forms in the experimental group increased by 1.6, 2.0, 1.6, and 1.5 times respectively.

[0047] It shows that during the process of Pseudomonas stutzeri biosynthesizing calcium carbonate precipitate using calcium acetate and calcium citrate by the repair method provided by the present invention, lead ions in the soil can be precipitated in the form of coprecipitation or locked in the crystal lattice of calcium carbonate, thereby effectively reducing the mobility and toxicity of soil lead and achieving the purpose of in-situ passivation of soil lead and safe utilization of soil.

[0048] 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 fields can still make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all technical solutions obtained by adopting equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A method for bioremediating lead - contaminated saline - alkali soil by biomineralization of Pseudomonas stutzeri, characterized in that, Pseudomonas stutzeri that has passed the activity test and adaptability test is used as a remediation bacterium agent, calcium acetate and calcium citrate are used as calcium sources, and the remediation bacterium agent, calcium acetate, calcium citrate, urea, and potassium dihydrogen phosphate are dissolved in water to form a soil remediation solution; the soil remediation solution is applied to the soil to be remediated, and the soil is maintained in an aerobic environment; Pseudomonas stutzeri degrades the calcium source to biosynthesize calcium carbonate precipitate, and during the biosynthesis process, lead ions in the soil are precipitated or locked in the calcium carbonate lattice in the form of coprecipitation, thereby reducing the mobility and toxicity of lead in the soil and achieving the purpose of in-situ remediation of lead pollution in saline soil by biomineralization.

2. The method for bioremediating lead-contaminated saline soil by biomineralization using Pseudomonas stutzeri according to claim 1, characterized in that, The Pseudomonas stutzeri is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC KB20081858.

3. The method for bioremediating lead-contaminated saline soil by biomineralization using Pseudomonas stutzeri according to claim 1, characterized in that The soil remediation solution contains 0.01 - 0.05 g / L of Pseudomonas stutzeri powder, 1 - 5 g / L of calcium acetate, 1 - 5 g / L of calcium citrate, 0.4 - 2 g / L of urea, and 0.05 - 0.25 g / L of potassium dihydrogen phosphate.

4. The method for bioremediating lead-contaminated saline soil by biomineralization using Pseudomonas stutzeri according to claim 1, characterized in that The specific methods of the activity test and adaptability test are as follows: Disperse Pseudomonas stutzeri into physiological saline to obtain an initial bacterial solution; inoculate the initial bacterial solution into a first culture medium for growth activity culture; detect the OD 600 value, and take the first bacterial solution with an OD 600 value greater than 1.5 to complete the growth activity detection; The bacterial solution that has completed the growth activity detection is inoculated into a second culture medium for constant temperature culture; When the OD of the bacterial solution in the second culture medium 600 value is greater than 1.5, take a small amount of the precipitate, wash it, and add a hydrochloric acid solution with a concentration of 1 M for adaptability test; if bubbles appear in the precipitate, the adaptability test is passed; The first culture medium includes 2.5 g / L of calcium acetate, 2.5 g / L of calcium citrate, 1.0 g / L of urea, 0.2 g / L of potassium dihydrogen phosphate, and 5.0 g / L of sodium chloride. The preparation method of the second culture medium is as follows: 2.5 g / L of calcium acetate, 2.5 g / L of calcium citrate, 1.0 g / L of urea, and 0.2 g / L of potassium dihydrogen phosphate are added to the soil solution and then filtered through a 0.45 μm filter membrane.

5. The method for bioremediating lead-contaminated saline soil by biomineralization using Pseudomonas stutzeri according to claim 4, characterized in that, The mass concentration of Pseudomonas stutzeri in the initial bacterial solution is 2 g / L; the inoculation amount of the initial bacterial solution in the first culture medium is 1%; the inoculation amount of the bacterial solution in the second culture medium is 1%.

6. The method for bioremediating lead-contaminated saline soil by biomineralization using Pseudomonas stutzeri according to claim 4, characterized in that, The preparation method of the soil solution is as follows: the soil to be remediated that has passed through a 100-mesh sieve is dispersed in water at a mass concentration of 100 g / L to obtain a soil mixture; the soil mixture is oscillated for 8 hours at 30°C and 170 rpm and then filtered through a 0.45 μm filter membrane to obtain the supernatant, which is the soil solution.

7. The method for repairing lead-contaminated saline soil by biomineralization using Pseudomonas stutzeri according to claim 1, characterized in that, The EC value of the soluble salt concentration in the soil to be remediated is less than 50 dS / m, the soil pH range is 5 - 8, the depth of soil lead pollution is less than 40 cm, and the soil lead concentration is less than 100 mg / kg.

8. The method for repairing lead-contaminated saline soil by biomineralization using Pseudomonas stutzeri according to claim 1, characterized in that The application amount of the soil remediation solution is 1 - 4 L per square meter of soil.

9. The method for bioremediating lead-contaminated saline soil by biomineralization using Pseudomonas stutzeri according to claim 1, characterized in that, The soil to be remediated is plowed with a rotary tiller, and the soil remediation solution is sprayed while plowing; the number of times of plowing and spraying the soil remediation solution is 2 - 3 times.

10. The method for bioremediating lead-contaminated saline soil by biomineralization using Pseudomonas stutzeri according to claim 1, wherein, The temperature maintained during the remediation process of the soil to be remediated is 10 - 35°C, the soil moisture content is 10 - 30%, and the remediation cycle is 5 - 10 days.