A method for repairing cadmium contaminated soil

Abstract

The present application belongs to the field of soil remediation, and particularly relates to a method for remediation of cadmium contaminated soil. The specific technical scheme is as follows: a soil remediation method comprises the following steps: before planting economic crops, applying sulfate-reducing bacteria to the soil to be remediated; after harvesting the economic crops, planting cadmium extraction crops in the soil to be remediated. The present application provides a water-flood rotation soil remediation method which can produce while remediation. On the one hand, the biological passivation can reduce the biological availability of cadmium in the soil, thereby reducing the stress of cadmium on plants and the absorption and accumulation of cadmium by plants; on the other hand, the biological technology can strengthen the ability of nightshade to extract cadmium, thereby extracting cadmium in farmland soil.

Description

Technical Field

[0001] This invention belongs to the field of soil remediation, and specifically relates to a method for remediating cadmium-contaminated soil. Background Technology

[0002] Cadmium pollution in soil has become a global problem. Compared with other trace elements, cadmium in soil is more mobile and more easily absorbed by crop roots, then transferred to the edible parts of crops and enters the human body through the food chain. Therefore, when farmland is contaminated with cadmium, regulating the transfer and accumulation behavior of the soil-crop system is key to preventing cadmium pollution from affecting human health.

[0003] Among methods for remediating cadmium-contaminated soil, passivation remediation is an effective approach that does not disrupt agricultural production. However, this method cannot fundamentally remove cadmium from the soil, and stable cadmium in the soil may be reactivated and released when environmental conditions change. While phytoremediation can completely remediate cadmium pollution in the soil without secondary pollution, its remediation process is lengthy, and the extended remediation period cannot guarantee the output and income of farmland.

[0004] In summary, it would be of great significance to develop a remediation technology that can thoroughly restore soil cadmium pollution without affecting farmland production, based on the concept of "remediation while production is carried out" and taking into account the characteristics of heavy metal cadmium during crop rotation, and relying on biopassivation and plant extraction technologies. Summary of the Invention

[0005] The purpose of this invention is to provide a method for remediating cadmium-contaminated soil.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is: a soil remediation method, comprising the following steps:

[0007] (1) Apply sulfate-reducing bacteria to the soil to be restored before planting cash crops;

[0008] (2) After the cash crops are harvested, cadmium extraction crops are planted in the soil to be restored.

[0009] Preferably, the screening method for sulfate-reducing bacteria in step (1) is as follows: FeSO4 is added to the microbial culture medium, and black colonies in the culture medium are selected.

[0010] Preferably, the sulfate-reducing bacteria in step (1) is Desulfovibriovulgaris SRB-CIB, which was deposited at the China General Microbiological Culture Collection Center on June 4, 2018, with accession number CGMCC NO.15859.

[0011] Preferably, the cadmium-extracting crop is a crop grown in dryland.

[0012] Preferably, the cadmium-extracting crop is black nightshade.

[0013] Preferably, the cash crop is a crop grown in paddy fields.

[0014] Preferably, the cash crop is rice.

[0015] Preferably, after the cash crops are harvested, the soil moisture is drained, the soil is turned over and dried, and then cadmium extraction crops are planted.

[0016] Preferably, after turning and drying the soil, microorganisms that increase the bioavailability of cadmium are applied to the soil before planting cadmium-extracting crops.

[0017] Preferably, the microorganisms that increase cadmium bioavailability include ferrous oxidizing bacteria that can oxidize ferrous ions and sulfur oxidizing bacteria that can oxidize elemental sulfur and reduced sulfur.

[0018] This invention offers the following beneficial effects: It provides a method for soil remediation through crop rotation that combines production and restoration. On one hand, it reduces the bioavailability of cadmium in the soil through biological passivation, thereby reducing the stress of cadmium on plants and the accumulation of cadmium by plants; on the other hand, it enhances the ability of black nightshade to extract cadmium from farmland soil using biotechnology.

[0019] This invention allows for rice cultivation during paddy fields, reducing cadmium content in brown rice through biological passivation, thus ensuring safe rice production in lightly cadmium-contaminated farmland. After rice harvest, microorganisms are used to enhance the bioavailability of cadmium in the soil, while simultaneously strengthening the cadmium extraction effect of black nightshade, thereby reducing the total amount of cadmium in the soil. This crop rotation remediation model can thoroughly remediate soil cadmium pollution without affecting normal farmland output. This invention is simple to use, has significant economic benefits, and is suitable for large-scale application. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the principle of the method of the present invention;

[0021] Figure 2 This is an electron microscope scan of SRB-CIB. Detailed Implementation

[0022] This invention provides a method for effectively remediating cadmium-contaminated land / soil, such as... Figure 1 As shown, the main steps include the following:

[0023] 1. Before planting crops, apply sulfate-reducing bacteria to the contaminated land / soil. These bacteria transfer electrons from organic matter to SO4. 2- to convert it into S 2-This leads to the formation of the poorly soluble compound CdS with cadmium, reducing the bioavailability of cadmium in the soil and decreasing the absorption and accumulation of cadmium by crops. The sulfate-reducing bacteria is preferably *Desulfovibrio vulgaris* SRB-CIB, deposited on June 4, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 15859. The 16S rDNA of *Desulfovibrio vulgaris* is shown in SEQ ID NO: 1.

[0024] 2. After harvesting crops, drain the water, turn the soil over and let it dry to increase the air in the soil and oxidize some of the reducing substances in the soil.

[0025] 3. Based on this, supplement with lysing-promoting bacteria and simultaneously plant black nightshade. This will accelerate the soil oxidation rate, oxidizing CdS to SO4. 2- and Cd 2+ This process simultaneously lowers soil pH, activating and dissolving cadmium in the soil; it also facilitates the transfer of cadmium from the soil to the black nightshade spores, effectively reducing the total amount of cadmium in the soil by harvesting the black nightshade. Specific lysis-promoting bacteria can further enhance the absorption of cadmium by the black nightshade, further reducing the cadmium content in the soil. These lysis-promoting bacteria can oxidize elemental sulfur and reduced sulfur in the soil. A preferred embodiment includes *Sulfobacillus*, *Phenylobacterium*, and *Ralstonia*.

[0026] Under the above-mentioned crop rotation model, the combination of land production and restoration can be effectively achieved.

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. All obtained data are average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0028] The culture media and reagents involved in this invention are as follows:

[0029] 1. Solid purification medium: KH2PO4 0.5g / L, NH4Cl 1.0g / L, CaCl2·2H2O 0.1g / L, MgSO4·7H2O 2.0g / L, yeast extract 1.0g / L, sodium lactate (70%) 5mL / L, Na2SO4 1.0g / L, agar 20g / L. Autoclave at 121℃ for 20min, then cool to about 50℃. Add filtered and sterilized FeSO4·7H2O 1.5g / L, VC 0.1g / L, sodium thioglycolate 0.1g / L, and L-Cys cysteine ​​0.5g / L. Adjust the pH to 8.0 with constant stirring.

[0030] 2. Liquid culture medium A: KH₂PO₄ 0.5g, NH₄Cl 1.0g, CaCl₂·2H₂O 0.1g, Na₂SO₄ 1.0g, MgSO₄·7H₂O 2.0g, sodium lactate (70%) 15mL, yeast extract 8.0g, distilled water 1000mL. Sterilize at 121℃ for 20min, then add FeSO₄·7H₂O 15g, ascorbic acid 0.1g, sodium thioglycolate 0.1g, cysteine ​​0.5g. Filter through a 0.22μm PES membrane for sterilization, and adjust the pH to 8.0–8.5.

[0031] 3. Liquid Culture Medium B: KH₂PO₄ 0.5g, NH₄Cl 1.0g, CaCl₂·2H₂O 0.1g, Na₂SO₄ 1.0g, MgSO₄·7H₂O 2.0g, sodium lactate (70%) 25mL, yeast extract 5.0g, distilled water 1000mL. Sterilize at 121℃ for 20min, then add FeSO₄·7H₂O 15g, ascorbic acid 0.1g, sodium thioglycolate 0.1g, cysteine ​​0.5g. Filter through a 0.22μm PES membrane for sterilization, and adjust the pH to 8.0–8.5.

[0032] 4. Starky liquid medium: (NH4)2SO4 3g, KCl 0.1g, K2HPO4 3g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, CaCl2·2H2O 0.25g, S 10g, distilled water 1000mL, pH adjusted to 2.5 with H2SO4. Elemental S, sterilized by UV irradiation, is mixed with the remaining portion of the autoclaved medium.

[0033] 5. 9K liquid culture medium: (NH4)2SO4 3g, KCl 0.1g, K2HPO4 0.5g, MgSO4·7H2O 0.5g, Ca(NO3)2 0.01g, FeSO4·7H2O 44.7g (filtered for sterilization), distilled water 1000mL, pH adjusted to 2.0 with H2SO4.

[0034] Example 1: Screening and Identification of Microorganisms

[0035] 1. Screening and Identification of Sulfate-Reducing Bacteria. Sulfate-reducing bacteria producing high levels of sulfides were screened from sulfate-reducing microorganisms preserved at the Chengdu Institute of Biology, Chinese Academy of Sciences. The method was as follows: 100 μL of each microorganism's bacterial suspension was drawn using a disposable sterile syringe and spread onto a solid purification medium. The medium was placed in an anaerobic chamber, with an anaerobic bag added to absorb oxygen. The chamber was then incubated at 35°C for 4 days. Darker single colonies were selected and streaked onto fresh solid purification medium. After another 4 days of incubation at 35°C, the culture was removed. After repeated streaking purification, sulfate-reducing bacteria SRB1, SRB2, SRB3, and SRB-CIB were obtained. The reason for selecting darker single colonies is that with the addition of FeSO4·7H2O to the medium, sulfate-reducing bacteria can reduce sulfate ions to sulfide ions. Sulfide ions react with ferrous ions to form black FeS. Therefore, the colonies of sulfate-reducing bacteria appear black in the medium, and the darker the colony, the more sulfides it produces.

[0036] Liquid culture medium A contained 8 g / L yeast extract and 15 mL / L sodium lactate as carbon and nitrogen sources, while liquid culture medium B contained 5 g / L yeast extract and 25 mL / L sodium lactate as carbon and nitrogen sources. Sulfate-reducing bacteria SRB1, SRB2, SRB3, and SRB-CIB were inoculated at 10% (v / v) into liquid culture media A and B, respectively, and cultured anaerobically at 35°C for 12 days. The sulfide content in each culture medium was determined using the methylene blue spectrophotometric method (GB / T16489-1996), and high-sulfate-producing sulfate-reducing bacteria were screened. The results are shown in Table 1.

[0037] Table 1. Sulfide production by different sulfate-reducing bacteria in different culture media.

[0038] strain Liquid culture medium A Liquid culture medium B SRB1 1275mg / L 1674mg / L SRB2 1216mg / L 1708mg / L SRB3 1152mg / L 1656mg / L SRB-CIB 1271mg / L 1810mg / L

[0039] SRB-CIB produced the highest overall sulfide content, so SRB-CIB was selected for subsequent tests.

[0040] Upon identification, the physiological and biochemical characteristics of SRB-CIB are as follows: SRB-CIB is a Gram-negative bacterium with black colonies, a raised surface, rod-shaped cells that are slightly curved and blunt at both ends. Its scanning electron microscope image is shown below. Figure 2As shown in the figure. 16S rDNA of SRB-CIB was extracted, as shown in SEQ ID NO: 1. Upon comparison, it showed a 99% similarity to *Desulfovibrio vulgaris*, and was identified as *Desulfovibrio vulgaris*. The *Desulfovibrio vulgaris* SRB-CIB was deposited on June 4, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 15859.

[0041] 2. Screening and identification of lysing-promoting bacteria. Naturally acidic (pH < 7) soil from a farmland in Wuhou District, Chengdu City was selected. 5g of soil sample was added to 250mL Erlenmeyer flasks containing 100mL 9K medium or 100mL Starky medium and cultured at 30℃ and 160r / min for 7–10 days.

[0042] For 9K medium, select the group where the solution is turbid, the medium is red, and there is ferric precipitation; this group contains ferrous oxidizing bacteria. For Starky medium, select the group where the medium is uniformly turbid, sulfur particles settle in the culture system, and the pH drops to 1; this group contains sulfur oxidizing bacteria.

[0043] The ferrous oxidizing bacteria were identified as primarily *Sulfobacillus*, with a relative abundance of 82.64%. The sulfur-oxidizing bacteria mainly included *Phenylobacterium*, *Ralstonia*, and *Herbaspirillum*, with relative abundances of 29.58%, 26.08%, and 23.08%, respectively.

[0044] The same screening method was repeated 3 times, and the composition of ferrous oxide functional bacteria and sulfur oxide functional bacteria remained basically stable, with no change in the main microbial species.

[0045] Example 2: Demonstration of Soil Remediation Effects

[0046] 1. Prepare bacterial culture.

[0047] Commercially available common desulfurized Vibrio was used as a control. The common desulfurized Vibrio SRB-CIB and the commercially available common desulfurized Vibrio were inoculated into liquid culture medium B and cultured at 35°C until the viable cell concentration reached ≥1.0 × 10⁻⁶. 8 The concentration of CFU / mL was increased, and the culture medium was removed by centrifugation to obtain pure SRB-CIB bacterial solution and commercially available desulfurized Vibrio bacterial solution.

[0048] Ferrous oxidizing functional bacteria were inoculated into 9K medium, and sulfur oxidizing functional bacteria were inoculated into Starky medium. Both were cultured at 30℃ and 160 r / min, respectively, until the viable cell concentration reached ≥7.5 × 10⁻⁶. 7 The culture medium was centrifuged to remove the CFU / mL concentration, yielding ferrous oxide functional bacteria suspension and sulfur oxide functional bacteria suspension. The ferrous oxide functional bacteria suspension and sulfur oxide functional bacteria suspension were then mixed at a volume ratio of 1:1 to obtain a lysis-promoting functional bacteria suspension.

[0049] Round plastic buckets (24.0 cm in diameter, 23.5 cm in height) were selected, and 5 kg of air-dried soil was placed in each bucket. In this embodiment, the DTPA extraction method (soil:DTPA = 1:5, w / w) was used to detect the available Cd content in the soil. The initial Cd content in the soil was 5 mg / kg, and the initial available Cd content was 1.8 mg / kg. 200 mL of the prepared SRB-CIB bacterial solution was resuspended in 200 mL of ultrapure water, and the solution was fully dissolved using a magnetic stirrer to obtain 400 mL of bacterial solution. This solution was then sprayed onto the soil and thoroughly mixed. Each group of soil was then completely submerged in water and allowed to stand for 5 days. Subsequently, base fertilizer was applied to the soil: N: 0.30 g / kg, P: 0.20 g / kg, K: 0.20 g / kg, in the forms of urea, superphosphate, and potassium chloride, respectively. Rice was planted after fertilization. Rice seedlings of roughly the same size were transplanted at a planting density of 4 plants per pot. Three groups were set up: an experimental group (treated with a standard desulfurized Vibrio spp. SRB-CIB bacterial solution), a positive control group (treated with an equal volume of commercially available standard desulfurized Vibrio spp. bacterial solution), and a blank control group (treated with an equal volume of ultrapure water). Except for the bacterial solution used, all other conditions were identical across groups, with 6 replicates per group. The potted plants were placed in a row, with their positions randomly rotated at irregular intervals. Throughout the growth period, the plants were frequently irrigated with shallow water to simulate a paddy field flooded state (water level 3–6 cm).

[0050] After the rice reached full maturity, mature rice plants from each group were harvested, and the cadmium content in four parts—brown rice, husk, stems and leaves, and roots—was measured. Simultaneously, the rhizosphere soil of the rice was collected to determine the content of available cadmium (DTPA). The results are shown in Table 2, with cadmium content expressed in mg / kg.

[0051] Table 2 Comparison of cadmium content in each group

[0052]

[0053] After the rice harvest, the soil from the six replicate pots in each group was poured out, thoroughly mixed, and allowed to air dry naturally for 20 days before being repackaged into six pots for planting black nightshade. Before planting, the experimental group was supplemented with 400 mL of lysing-promoting bacterial solution (400 mL of lysing-promoting bacterial solution was resuspended in 400 mL of ultrapure water); the control group was supplemented with an equal volume of ultrapure water. All treatments were thoroughly mixed after application.

[0054] Two black nightshade seedlings were transplanted into each pot, with no significant differences among the seedlings. Soil moisture content was maintained at 50%–80% of field capacity during the black nightshade's growth period. Black nightshade was harvested after 60 days of growth. Harvested plant samples were divided into root and aboveground parts. The roots and aboveground parts were thoroughly rinsed with tap water to remove adhering soil and debris, then rinsed with pure water and drained. The plant samples were then placed in envelopes, blanched at 110℃ for 30 minutes, and dried at 70℃ to constant weight. The dried samples were pulverized into powder, passed through a 60-mesh sieve, and dried again. The available cadmium content was measured, and the cadmium accumulation in the whole black nightshade plant was determined for each group. The cadmium content in plant tissues was determined using nitric acid-hydrogen peroxide microwave digestion-graphite furnace atomic absorption spectrophotometry. Specifically, 0.1 g of plant tissue sample was weighed and placed in a polytetrafluoroethylene digestion vessel. 5 mL of analytical grade concentrated nitric acid and 30% hydrogen peroxide were added, and the mixture was then placed in a microwave digester for digestion (150℃, 25 min). The digested samples were diluted to 100 mL with ultrapure water in a glass volumetric flask, filtered, and the Cd concentration was determined using a graphite furnace atomic absorption spectrophotometer (AAS). The cadmium accumulation in *Solanum nigrum* plants was calculated as: plant biomass (dry weight) × total cadmium concentration in the plant. Simultaneously, rhizosphere soil samples from each group were collected and sieved through a 100-mesh sieve to determine the available cadmium content. The results were averaged from replicates. The results are shown in Table 3.

[0055] Table 3 Comparison of cadmium content in each group

[0056]

[0057] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A soil remediation method, characterized in that: Includes the following steps: (1) Before planting economic crops, sulfate-reducing bacteria are applied to the soil to be remediated; the sulfate-reducing bacteria is Desulfovibrio vulgaris SRB-CIB, which was deposited at the China General Microbiological Culture Collection Center on June 4, 2018, with the accession number: CGMCC NO.15859. (2) After the harvest of cash crops, the water in the soil is drained, the soil is turned over and dried, and microorganisms that increase the bioavailability of cadmium are applied to the soil. Then, cadmium-extracting crops are planted in the soil to be remediated. The cadmium-extracting crops are black nightshade, and the microorganisms that increase the bioavailability of cadmium include Sulfobacillus, Phenylobacterium and Ralstonia.

2. The method according to claim 1, characterized in that: The cash crops mentioned are those grown in paddy fields.

3. The method according to claim 2, characterized in that: The cash crop mentioned is rice.

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