A method for micro-bioremediation of cadmium-contaminated soil

Abstract

This invention relates to the field of remediation and restoration technology for cadmium-contaminated soil, specifically a method for microbial remediation of cadmium-contaminated soil. The method includes the following steps: Step 1: Cultivating *Tetrahymena thermophila* to the logarithmic growth phase to obtain a *Tetrahymena thermophila* culture medium; Step 2: Repeatedly soaking the cadmium-contaminated soil in clean water, rinsing with a leaching agent, then allowing it to stand, separating, and collecting the leaching solution, which is then adjusted to neutral; Step 3: Centrifuging the *Tetrahymena thermophila* culture medium to obtain *Tetrahymena thermophila*; inoculating the *Tetrahymena thermophila* into the leaching solution, allowing it to stand for a period of time to obtain a cadmium-removed solution; finally, separating and collecting the *Tetrahymena thermophila* and the overlying water to complete the remediation. This invention is simple to operate, requires minimal investment, and has a short remediation time. Furthermore, it possesses the ability to thoroughly remediate cadmium-contaminated soil, thus having broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of treatment and remediation technology for soil contaminated with heavy metal cadmium, specifically a method for micro-bioremediation of cadmium-contaminated soil. Background Technology

[0002] Soil is a vital component of the ecosystem and an indispensable foundation for crop growth. However, heavy metal pollution in soil can lead to reduced food yields, and crops have a strong ability to accumulate heavy metals. Ultimately, these heavy metals enter the human body through the food chain, causing harm. Cadmium (Cd) is a common heavy metal found in soils such as paddy fields, and rice has a strong ability to accumulate cadmium. Long-term consumption of cadmium-contaminated rice can cause diseases such as Itai-itai disease and kidney failure. Therefore, removing cadmium from contaminated soils such as paddy fields is of great significance for ensuring agricultural food safety.

[0003] Currently, there are many methods for remediating cadmium-contaminated soil, such as physical, chemical, and bioremediation methods. Examples include in-situ remediation using passivating agents (Chinese Patent Publication No. CN110283596A), soil remediation using Bacillus subtilis (Chinese Patent Publication No. CN202310729331), phytoremediation of cadmium-contaminated soil (Chinese Patent Publication No. CN109304366A), and even composite schemes combining microbial agents and phytoremediation (Chinese Patent Publication No. CN110252801A). However, these remediation methods often suffer from difficulties in implementation, high costs, long remediation cycles, low remediation efficiency, the risk of secondary pollution, and incomplete cadmium removal. Among these, bioremediation has lower energy consumption and is relatively environmentally friendly; however, existing remediation processes often rely on in-situ addition or planting, which makes it difficult to fully realize the advantages of bioremediation and achieve complete remediation of cadmium-contaminated soil.

[0004] Considering that the key to the complete remediation of cadmium-contaminated soil lies in the effective removal of cadmium, Tetrahymena thermophila, a single-celled organism, has a strong adsorption capacity for cadmium. It can absorb most of the cadmium in the soil in a short time and convert it into CdS nanoparticles intracellularly to reduce the bioavailability of cadmium. Furthermore, Tetrahymena thermophila has the characteristics of rapid growth, simple cultivation, and low cost. Therefore, the application of Tetrahymena thermophila to completely remediate cadmium-contaminated soil has great potential.

[0005] In summary, this invention combines biological cadmium removal and other technologies to propose a method for remediating cadmium-contaminated soil using thermophilic tetrahymena, which can thoroughly remediate cadmium-contaminated soil while avoiding secondary pollution. Summary of the Invention

[0006] The purpose of this invention is to address the problem that existing methods for remediating cadmium-contaminated soil mainly rely on chemical and physical methods, which are insufficient to fundamentally separate cadmium from the soil. Therefore, this invention provides a method for the complete remediation of cadmium-contaminated soil using microorganisms.

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

[0008] A method for remediating cadmium-contaminated soil using microorganisms, characterized in that: microorganisms are used to remediate cadmium-contaminated soil; wherein the microorganisms are thermophilic tetrahymena.

[0009] Preferably, the thermophilic tetrahymena includes thermophilic tetrahymena SB210 or its improved strain.

[0010] A preferred embodiment of the method for microbial remediation of cadmium-contaminated soil includes the following steps:

[0011] Step 1: Cultivating Tetrahymena thermophila: Inoculate Tetrahymena thermophila into SPP medium, add 1% penicillin-streptomycin (100 U / mL penicillin and 100 μg / mL streptomycin), seal the bottle, and incubate at 20-35℃ for 1-2 days. Then transfer to a constant temperature shaker incubator for amplification culture to obtain Tetrahymena thermophila culture medium.

[0012] Step 2: Acidification and leaching of cadmium-contaminated soil to promote cadmium release: (1) Soak cadmium-contaminated soil in clean water and add leaching agent to adjust the pH to 2-4. After standing for 12-48 hours, separate the leaching solution and adjust the pH of the leaching solution to neutral for biological cadmium removal; (2) Repeat the operation in (1) on the cadmium-contaminated soil obtained in (1) and collect the leaching solution until the cadmium concentration in the final cadmium-contaminated soil is lower than the standard value of 0.3 mg / kg of the "Soil Environmental Quality Agricultural Land Soil Pollution Risk Control Standard" and the cadmium concentration in the last leaching solution is lower than the Class II water standard value of 5 μg / L of the "Surface Water Environmental Quality Standard". Then add limestone to restore the pH of the soil with cadmium content that meets the standard.

[0013] Step 3: Cadmium removal treatment: (1) Centrifuge the Tetrahymena thermophila culture medium at a speed of 1000-2000 rpm for 2-10 minutes, remove the supernatant, collect the precipitate, and obtain Tetrahymena thermophila; (2) Inoculate Tetrahymena thermophila into the rinsing solution and place it at room temperature for 12-96 hours to obtain cadmium removal solution; (3) Separate and collect Tetrahymena thermophila and overlying water from the cadmium removal solution, perform harmless sealing treatment on Tetrahymena thermophila, and then test the overlying water. When the cadmium concentration in the overlying water meets the relevant national standards and is lower than the Class II water standard value of 5 μg / L in the "Surface Water Environmental Quality Standard", the overlying water is naturally discharged or recycled to complete the remediation.

[0014] Preferably, the SPP culture medium is formulated with the following concentration components: peptone 15-25 g / L, yeast extract 0.5-1.5 g / L, glucose 1-3 g / L, ferric citrate 0.01-0.05 g / L, and ultrapure water to a final volume of 1 L.

[0015] Preferably, the SPP culture medium is sterilized after preparation; the sterilization parameters are: sterilize at 100-105 kPa and 115-125°C for 10-30 minutes, and then cool to room temperature before storage.

[0016] Preferably, the parameters of the constant temperature shaking incubator are: incubation temperature 20-35℃, rotation speed 100-150 rpm, and incubation time 1-5 days.

[0017] Preferably, the density of Tetrahymena thermophila in the Tetrahymena thermophila culture medium is 0.5–1.5 × 10⁻⁶. 6 per milliliter.

[0018] Preferably, the rinsing agent includes, but is not limited to, any one of inorganic acid solutions and inorganic salt solutions; including but not limited to concentrated hydrochloric acid solution, concentrated sulfuric acid solution, concentrated nitric acid solution, ammonium sulfate solution, etc.

[0019] Preferably, in step three, the inoculum concentration of Tetrahymena thermophila in the eluent is 1×10⁻⁶. 5 ~1.5×10 6 per milliliter.

[0020] Preferredly, in step three, the separation method in (3) includes, but is not limited to, any one of coagulation sedimentation, flocculation sedimentation, and membrane filtration.

[0021] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0022] (1) In this invention, by culturing Tetrahymena thermophila to the logarithmic growth phase, a sufficient amount of microorganisms is obtained, and the high activity of Tetrahymena thermophila at this time is utilized to make cadmium removal more efficient.

[0023] (2) The present invention adjusts the soil pH to acidic by using a leaching agent, promotes the conversion of various forms of cadmium in cadmium-contaminated soil into acid-soluble cadmium, and collects leaching liquid containing cadmium, thereby increasing the contact amount of thermophilic tetrahymena with cadmium and effectively improving the removal rate of cadmium by thermophilic tetrahymena.

[0024] (3) In this invention, cadmium-contaminated soil can be repeatedly leached until the concentration of cadmium in the soil reaches the standard specified in GB15618-2018 "Soil Environmental Quality Agricultural Land Soil Pollution Risk Control Standard" (≤0.3mg / kg), so that cadmium in the cadmium-contaminated soil can be fully released into the leaching solution, thereby achieving the purpose of thoroughly remediating cadmium-contaminated soil.

[0025] (4) After cadmium removal treatment, the concentration of cadmium in the overlying water reaches the Class II water standard (≤5μg / L) specified in GB 3838-2002 "Surface Water Environmental Quality Standard". It can be directly discharged into the natural environment or reused, thus saving water resources.

[0026] (5) This invention has the advantages of simple operation process, low investment and short repair time, and has a high cadmium removal effect and broad application prospects. Attached Figure Description

[0027] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0028] Figure 1 To repair the process flow diagram;

[0029] Figure 2 The release of cadmium from soil under different acid treatments (pH=3);

[0030] Figure 3 The content of different forms of cadmium in the soil before and after leaching;

[0031] Figure 4 This is due to the cyclical release of cadmium from the soil.

[0032] Figure 5 Survival curves of Tetrahymena at different inoculation concentrations;

[0033] Figure 6 The inoculation concentration was 1.5 × 10⁻⁶. 6 Survival curve of Tetrahymena under the condition of cells / mL;

[0034] Figure 7 This refers to the cadmium concentration in the overlying water.

[0035] Figure 8 This represents the cadmium content within Tetrahymena. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] In this embodiment, it should be noted that there are no special restrictions on the manufacturers of the raw materials involved in this invention; exemplarily, they include:

[0038] The cadmium-contaminated soil came from Yanguantian in Yiyang County, Jiangxi Province, with a total cadmium concentration of 0.43 mg / kg.

[0039] Peptone, yeast extract, and glucose were all purchased from Thermo Scientific.

[0040] Ferric citrate, purchased from MilliporeSigma;

[0041] HCl solution, HNO3 solution, H2SO4 solution, HOAC solution, and (NH4)SO4 solution, 2 mol / L, were all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0042] Experimental protocol: A method for micro-bioremediation of cadmium-contaminated soil, comprising the following steps:

[0043] Step 1: 1. Prepare SPP medium: 20 g / L peptone, 1 g / L yeast extract, 2 g / L glucose, 0.03 g / L ferric citrate, and bring the volume to 1 L with ultrapure water; then sterilize at 103 kPa and 120 °C for 20 minutes, and after cooling to room temperature, obtain SPP medium;

[0044] 2. Cultivation of Tetrahymena thermophila: (1) Inoculate Tetrahymena thermophila SB210 into a container containing SPP medium, add 1% penicillin-streptomycin, seal the bottle, and incubate at 30℃ for 2 days; (2) Place the Tetrahymena thermophila cultured in (1) in a constant temperature shaker incubator for amplification culture (set the parameters of the constant temperature shaker incubator: culture temperature 30℃, rotation speed 135 rpm, culture time 1-5 days) to obtain Tetrahymena thermophila culture medium;

[0045] Step 2: Acidification and leaching of cadmium-contaminated soil to promote cadmium release: (1) Weigh 10kg of cadmium-contaminated soil and place it in a container. Pour in 20L of clean water and mix well. Then, leach the cadmium-contaminated soil with 2mol / L leaching agent and adjust the pH to 3. After thorough mixing, let it stand for 24 hours, separate, and obtain the leaching solution. Adjust the pH of the leaching solution to neutral and use it for biological cadmium removal. (2) Repeat the operation in (1) on the cadmium-contaminated soil obtained in (1). After cyclic leaching, collect the leaching solution until the concentration of cadmium in the cadmium-contaminated soil is lower than the standard value of 0.3mg / kg of the "Soil Environmental Quality Agricultural Land Soil Pollution Risk Control Standard" and the concentration of cadmium in the last leaching solution is lower than the Class II water standard value of 5μg / L of the "Surface Water Environmental Quality Standard". Finally, use limestone to restore the pH of the soil with cadmium content that meets the standard.

[0046] Step 3: Cadmium removal treatment: (1) Centrifuge the Tetrahymena thermophila culture medium at 1500 rpm for 3 minutes, remove the supernatant, collect the precipitate, and obtain Tetrahymena thermophila; (2) Inoculate Tetrahymena thermophila into the rinsing solution and place it at room temperature for 96 hours to obtain cadmium removal solution; (3) Separate and collect Tetrahymena thermophila and overlying water from the cadmium removal solution, perform harmless sealing treatment on Tetrahymena thermophila, and then test the overlying water. After the cadmium concentration in the overlying water reaches the relevant national standards (lower than the Class II water standard value of 5 μg / L in the "Surface Water Environmental Quality Standard"), the overlying water is naturally discharged or recycled to complete the remediation.

[0047] 1. To investigate the cadmium release from cadmium-contaminated soil after leaching with different acid or salt solutions, HCl, HNO3, H2SO4, HOAC, (NH4)SO4, and plain water were used for 7 days of cyclic leaching. The daily cyclic release of cadmium from the contaminated soil was measured, and the results are as follows: Figure 2 As shown.

[0048] 2. This study investigated the effects of 9-day cyclic leaching of cadmium-contaminated soil with and without HCl solution, comparing the content of different forms of cadmium before and after leaching, and the daily cyclic release of cadmium from the soil. The results are as follows: Figure 3 , Figure 4 As shown;

[0049] The results showed that leaching of cadmium-contaminated soil with HCl solution could maximize the release of cadmium.

[0050] 3. To investigate the effects of inoculating different concentrations (1×10⁻⁶) of the eluent obtained from rinsing with HCl solution. 5 Cells / ml, 5×10 5 cells / ml, 1.5×10 6 The survival of Tetrahymena thermophila at different time points was analyzed using samples of Tetrahymena thermophila (number per milliliter). The results are as follows: Figure 5 As shown;

[0051] The results show that using 1.5×10 6 When Tetrahymena thermophila is inoculated into the rinsing solution at an inoculation concentration of 12 individuals / mL, the survival rate is best, therefore this inoculation concentration is the best choice for cadmium removal treatment.

[0052] 4. In step three, further steps are performed using HCl solution as the rinsing agent and the inoculation concentration of Tetrahymena thermophila is 1.5 × 10⁻⁶. 6 Under the condition of *Tetrahymena thermophila* concentration / mL, cadmium-contaminated soil remediation was carried out. Specifically, 3L of extracted soil leachate was poured into each 5L container. *Tetrahymena thermophila* SB210, cultured to the logarithmic growth phase and in good condition, was centrifuged at 1500 rpm for 3 minutes to remove the supernatant. The precipitate was collected and then inoculated into the leachate. Subsequently, samples of the cadmium-removed solution were taken at 0, 6, 12, 24, 36, 48, 72, and 96 hours to detect the survival curve of *Tetrahymena thermophila*, the concentration of cadmium in the overlying water, and the cadmium content within the *Tetrahymena thermophila*. Specific results are as follows: Figure 6 , Figure 7 , Figure 8 As shown;

[0053] The results showed that, with HCl solution as the rinsing agent and an inoculation concentration of 1.5 × 10⁻⁶, the optimal concentration of *Tetrahymena thermophila* was 1.5 × 10⁻⁶. 6 Under the condition of cadmium-contaminated soil remediation, Tetrahymena thermophila showed good survival ability and strong cadmium removal ability in the leachate; after only 48 hours, it was able to remove 90% of the cadmium ions in the leachate, and after 96 hours, the cadmium removal rate reached 98%, which is lower than the Class I water standard.

[0054] In summary, the method for remediating cadmium-contaminated soil provided by this invention has a high cadmium removal rate and shows a significant remediation effect. Therefore, the use of Tetrahymena thermophila to remediate cadmium-contaminated soil has great potential.

[0055] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0056] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for micro-bioremediation of cadmium-contaminated soil, characterized in that: Microorganisms were used to remediate cadmium-contaminated soil, specifically Tetrahymena thermophila, including Tetrahymena thermophila SB210 or its improved strains. The method includes the following steps: Step 1: Inoculate Tetrahymena thermophila into the culture medium, and then amplify the culture to obtain Tetrahymena thermophila culture medium; Step 2: Repeatedly soak the cadmium-contaminated soil with clean water, adjust the pH to 2-4 with a leaching agent, let it stand, separate, collect the leaching solution, and then adjust the leaching solution to neutral. Step 3: The thermophilic tetrahymena culture medium was centrifuged to obtain thermophilic tetrahymena; the thermophilic tetrahymena was then inoculated into the rinsing solution and left to stand for a period of time to complete the repair. Specifically, step one involves inoculating Tetrahymena thermophila into a culture medium, adding penicillin-streptomycin, sealing the bottle, and incubating it at 20-35°C for 1-2 days before transferring it to a constant temperature shaker incubator for amplification culture to obtain Tetrahymena thermophila culture medium. Step 2 is as follows: (1) Soak the cadmium-contaminated soil in clean water and add leaching agent to adjust the pH to 2-4. Let it stand and separate to obtain leaching solution. Adjust the pH of the leaching solution to neutral for biological cadmium removal. (2) Repeat the operation in (1) on the cadmium-contaminated soil obtained in (1) until the concentration of cadmium in the cadmium-contaminated soil is ≤0.3mg / kg and the concentration of cadmium in the last leaching solution is ≤5μg / L. Then restore the pH of the soil with the cadmium content that meets the standard. Step 3 is as follows: (1) Centrifuge the Tetrahymena thermophila culture medium, remove the supernatant, collect the precipitate, and obtain Tetrahymena thermophila; (2) Inoculate Tetrahymena thermophila into the rinsing solution and place it at room temperature to obtain the cadmium-removed solution; (3) Separate and collect the cadmium-removed solution to obtain Tetrahymena thermophila and the overlying water. After the test meets the standards, the Tetrahymena thermophila is sealed in a harmless manner, and the overlying water is discharged naturally or recycled to complete the remediation.

2. The method for micro-bioremediation of cadmium-contaminated soil according to claim 1, characterized in that: The parameters of the constant temperature shaking incubator are: culture temperature 20-35℃, rotation speed 100-150 rpm, and culture time 1-5 days.

3. The method for micro-bioremediation of cadmium-contaminated soil according to claim 1, characterized in that: The density of Tetrahymena thermophila in the culture medium was 0.5–1.5 × 10⁻⁶. 6 per milliliter.

4. The method for micro-bioremediation of cadmium-contaminated soil according to claim 1, characterized in that: The rinsing agent includes any one of inorganic acid solution and inorganic salt solution.

5. The method for micro-bioremediation of cadmium-contaminated soil according to claim 1, characterized in that: The inoculum concentration of Tetrahymena thermophila in the rinsing solution was 1×10⁻⁶. 5 ~1.5×10 6 (3) The separation method includes any one of coagulation sedimentation, flocculation sedimentation, and membrane filtration.

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