A method for regulating cadmium availability in soil by combining sphingobium and earthworms

Abstract

This invention belongs to the field of soil remediation technology, specifically disclosing a method for regulating soil cadmium availability through the combined use of *Sphingobacterium sphingosine monophosphate* and earthworms. The method involves selecting cadmium-contaminated soil, adjusting the soil moisture to 60% saturation, and stabilizing for 48 hours. Then, earthworm and *Sphingobacterium sphingosine monophosphate* solutions are added to the soil, and the mixture is cultured for at least 20 days. The earthworm inoculation density is 15 g / kg soil, and the *Sphingobacterium sphingosine monophosphate* solution is added at a rate of 5 ml / kg soil. Compared with existing technologies, this invention uses *Sphingobacterium sphingosine monophosphate* and earthworms in combination for cadmium-contaminated soil, ensuring the activity of *Sphingobacterium sphingosine monophosphate* in the soil. It also improves soil cadmium availability, promotes the absorption and removal of heavy metals by hyperaccumulating plants, thereby achieving soil remediation. It avoids the secondary pollution problems associated with traditional chemical remediation methods. Furthermore, by jointly regulating soil heavy metal availability through *Sphingobacterium sphingosine monophosphate* and earthworms, it enables site-specific and diversified remediation of heavy metal-contaminated soils.

Description

Technical Field

[0001] This invention relates to the field of soil remediation technology, and in particular to a method for regulating soil cadmium availability through the combined use of Sphingosine monophosphate and earthworms. Background Technology

[0002] Heavy metal pollution is the most serious problem. Heavy metals, especially cadmium, remain in the soil for extended periods, inhibiting microbial growth and reducing microbial diversity. Simultaneously, heavy metal stress disrupts plant physiological metabolism, reducing the root system's ability to absorb nutrients, leading to nutrient deficiencies and inhibiting plant growth. Furthermore, when heavy metals are absorbed by plants, they enter the human body through the food chain, causing diseases such as hypertension, cardiovascular disease, and bone pain. In severe cases, they can even cause a range of cancers including skin cancer, cervical cancer, and liver cancer.

[0003] Currently, the most common bioremediation technologies mainly focus on two directions: one is to passivate soil with organic conditioners to reduce the availability of heavy metals in the soil and avoid toxicity to crops, thereby achieving soil remediation; the other is to add inorganic activators to increase the availability of heavy metals in the soil and then use hyperaccumulating plants to absorb and remove heavy metals, thereby achieving soil remediation.

[0004] This application focuses on the second direction mentioned above. However, the passivation of organic conditioners inevitably introduces some inorganic components, posing a potential hazard to the soil; the potential hazard of inorganic activators to the soil is even greater. Therefore, current bioremediation methods for heavy metals in soil all introduce inorganic components, harming soil health.

[0005] Sphingosporobacter and earthworms, as naturally occurring soil organisms and microorganisms, have the potential to remediate heavy metals without causing potential harm to the soil. It is known that Sphingosporobacter can be used for cadmium passivation in soil. If Sphingosporobacter and earthworms can be added to cadmium-contaminated soil to improve the availability of heavy metals, and then hyperaccumulating plants can be used to absorb and remove the heavy metals, thereby achieving soil remediation, secondary soil pollution will undoubtedly be reduced. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for regulating soil cadmium availability through the combined use of Sphingosine monophosphate and earthworms.

[0007] To achieve the above objectives, the present invention is implemented according to the following technical solution:

[0008] A method for regulating soil cadmium availability through the combined use of Sphingomyelin and earthworms includes the following steps:

[0009] S1. Select cadmium-contaminated soil, adjust the soil moisture to 60% saturation moisture content, and stabilize for 48 hours;

[0010] S2. Then add earthworms and Sphingosine monoclonal antibody solution to the soil and incubate at 25°C for more than 20 days; the inoculation density of earthworms is 15g / kg soil; the amount of Sphingosine monoclonal antibody solution added is 5ml / kg soil.

[0011] Furthermore, in step S2, sphingomyelin-Bacillus solution is first added to the soil, and then earthworms are added.

[0012] Furthermore, the method for regulating soil cadmium availability through the combined use of Sphingosine monocytogenes and earthworms further includes:

[0013] S3. Repeat steps S1-S2 once or multiple times.

[0014] Furthermore, the preparation process of the *Sphingosine mononitrate* solution is as follows:

[0015] Sphingosine monoclonal antibodies were picked using an inoculation loop and streaked on a solid CM0827 Propionibacterium tibularum agar plate for isolation. The plates were incubated at 30°C until single colonies appeared. A single colony was then picked and inoculated into liquid CM0827 Propionibacterium tibularum agar plate. The pH of the medium was adjusted to 7.0, and the plates were incubated at a constant temperature of 30°C for 2 days until the OD405 value of the CM0827 Propionibacterium tibularum agar plate reached 1.45, yielding the Sphingosine monoclonal antibody solution. The solid CM0827 Propionibacterium tibularum agar plate consisted of the following components: The first nutrient solution consists of: 15g tryptone, 0.5g L-cysteine, 5g yeast extract, 0.5g sodium mercaptoglycolate, 5g glucose, 15g agar, 2.5g sodium chloride, 0.001g resazurin, and 1L distilled water. The second nutrient solution consists of: 15g tryptone, 0.5g L-cysteine, 5g yeast extract, 0.5g sodium mercaptoglycolate, 5g glucose, 0.7g agar, 2.5g sodium chloride, 0.001g resazurin, and 1L distilled water.

[0016] Preferably, the earthworm is a Moravian farbaceous worm.

[0017] Compared with existing technologies, this invention uses sphingosine monoclonal bacteria and earthworms in combination to treat cadmium-contaminated soil, which can ensure the activity of sphingosine monoclonal bacteria in the soil; at the same time, it can improve the cadmium availability in the soil, promote the absorption and removal of heavy metals by hyperaccumulating plants, and thus achieve soil remediation; it avoids the secondary pollution problems caused by traditional chemical remediation methods; in addition, by regulating the availability of heavy metals in the soil through the combined use of sphingosine monoclonal bacteria and earthworms, it is possible to remediate heavy metal soils in a diversified manner according to local conditions. Attached Figure Description

[0018] Figure 1 These are microscopic images and colony images of Sphingosine mononitrate.

[0019] Figure 2 Comparison of OD values ​​between Sphingosine monofilament solution and culture medium.

[0020] Figure 3 The growth curve of Sphingosine mononitrate is shown.

[0021] Figure 4 The growth curves of Sphingosine mononitrate under different cadmium concentrations are shown.

[0022] Figure 5 The content of cadmium in DTPA leaching state under different treatments.

[0023] Figure 6 This is a flowchart of the present invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0025] The sources of raw materials and reagents used in the following examples are:

[0026] Test strain: Sphingobacterium sp CICC 23244, purchased from the Beina Innovation Biotechnology Research Institute of China Microbial Strains Network.

[0027] Liquid culture medium: CM0827 Propionibacterium broth was used, with the following formula: 15g tryptone, 0.5g L-cysteine, 5g yeast extract, 0.5g sodium mercaptoglycolate, 5g glucose, 0.7g agar, 2.5g sodium chloride, 0.001g resazurin, and 1L distilled water.

[0028] Solid culture medium: Solid CM0827 Propionibacterium broth was used, with the following formula: 15g tryptone, 0.5g L-cysteine, 5g yeast extract, 0.5g sodium mercaptoglycolate, 5g glucose, 15g agar, 2.5g sodium chloride, 0.001g resazurin, and 1L distilled water.

[0029] Preparation of Sphingosine monofilament suspension: Sphingosine monofilaments were picked up using an inoculation loop and isolated by streaking on a solid CM0827 Propionibacterium tectonicus medium plate. The plate was cultured at 30°C until a single colony grew. A single colony was picked up using an inoculation loop and inoculated into liquid CM0827 Propionibacterium tectonicus medium. The pH of the medium was adjusted to 7.0, and the plate was cultured at a constant temperature of 30°C for 2 days until the OD405 value of the CM0827 Propionibacterium tectonicus medium was 1.45, thus obtaining the Sphingosine monofilament suspension.

[0030] Earthworm species used in this experiment: *Pheretima mouliensis* was selected as the earthworm species. *Pheretima mouliensis* is a South China earthworm species that feeds mainly on soil particles and organic matter mixtures. It generally lives in the 0-10cm soil layer and was collected from the campus of South China Agricultural University. After all earthworms were cultured for one week, healthy earthworms with mature rings or near-mature rings were selected for the experiment.

[0031] Test soil: The contaminated soil for this experiment was collected from a paddy field approximately 6 km downstream of the Dabao Mountain open-pit mining area in northern Guangdong Province (24°30′N, 113°45′E). The basic physicochemical properties of the soil were: pH: 4.40±0.03; organic carbon: 16.63±0.21 g·kg⁻¹. -1 Total nitrogen: 1.62 ± 0.09 g·kg -1 Clay content: 19.42±0.49%; Total cadmium: 0.64±0.20 mg·kg -1 .

[0032] Example 1: Cadmium resistance test of Sphingosine mononitrate bacteria

[0033] Sphingosine monoclonal antibodies were picked using an inoculation loop and isolated by streaking on a solid CM0827 Propionibacterium tartrate agar plate. The plates were incubated at 30°C until single colonies appeared. A single colony was then picked using an inoculation loop, washed with 5 ml of sterile water, and transferred to an Erlenmeyer flask containing 200 ml of liquid CM0827 Propionibacterium tartrate agar. The flask was incubated at 180 rpm and 30°C for 16 hours using a constant temperature shaker. Morphological observation of the strain was then performed: a small amount of the expanded culture was diluted, smeared, fixed, and Gram-stained. The smear was rinsed with sterile water, dried, and examined under a microscope.

[0034] Select the most suitable wavelength: Take liquid CM0827 Propionibacterium tumefaciens medium, inoculate it with Sphingosine monofilament suspension (the ratio of medium to bacterial suspension is 20:1), and incubate in a constant temperature shaker at 180 r / min and 30℃ for 24 h. Take it out and measure the absorbance at wavelengths of 405, 450, 490, 570, 600 and 630 nm using an ELISA reader (with uninoculated medium as a control).

[0035] To plot the growth curve: Take liquid CM0827 Propionibacterium tumefaciens medium and inoculate it with Sphingosine monofilamentum suspension (the ratio of medium to bacterial suspension is 20:1). Incubate in a constant temperature shaker at 180 r / min and 30℃. Take it out at 0, 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88 and 96 h and measure the absorbance value of the optimal wavelength using an ELISA reader (with uninoculated medium as a control).

[0036] Cadmium tolerance determination: Liquid CM0827 Propionibacterium culturae with different cadmium concentrations were prepared, resulting in final cadmium concentrations of 0, 0.25, 0.5, 1, 1.5, 3, 6, 12, 24, 48, 96, and 192 mg / L, with three replicates. Sphingosine monofiliis in the logarithmic growth phase were inoculated and cultured in a constant temperature shaker at 180 rpm and 30 °C. The cultures were collected at 0, 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, and 96 h, and the absorbance at the optimal wavelength was measured using a microplate reader (with uninoculated culture medium at the corresponding cadmium concentration used as a control).

[0037] Microscopic observation revealed that *Sphingospora* is a Gram-negative bacterium. *Sphingospora* is a long rod-shaped bacterium, approximately 0.1 μm × (0.5–0.8) μm in size. When *Sphingospora* was inoculated onto solid CM0827 Propionibacterium agar, the colonies were milky white, opaque, smooth, and had regular edges. Microscopic images and colony images of *Sphingospora* are shown below. Figure 1 .

[0038] Figure 2 This is a comparison of the OD values ​​of *Sphingosine monocytogenes* cultured for 24 hours at different wavelengths with the OD values ​​of the culture medium. Figure 2 It can be seen that the longer the wavelength, the lower the absorbance of both the culture medium and the bacterial suspension. At a wavelength of 405 nm, both the culture medium and the bacterial suspension have high absorbance, with a larger difference compared to other wavelengths. At 650 nm, although the absorbance of the culture medium is close to 0, the absorbance of the bacterial suspension is much lower, resulting in a smaller difference. If the absorbance of the culture medium is too high, most of the light beam transmitted to the bacterial suspension will be absorbed by the culture medium, affecting the absorbance of the bacteria and failing to accurately reflect their growth. Since the difference between the culture medium and the bacterial suspension measured at 405 nm is relatively large, it indicates that at a wavelength of 405 nm, the culture medium has a relatively small impact on the determination of the OD value of the bacterial suspension. Therefore, 405 nm was selected as the optimal absorption wavelength for *Sphingosine mononitrate*.

[0039] The growth curve of *Sphingosine mononitrate* measured at a wavelength of 405 nm is shown below. Figure 3 The growth curve of *Sphingosine monocytogenes* shows a lag phase of 0-8 hours, a logarithmic growth phase of 8-24 hours, a stationary phase of 24-88 hours, and a decline phase after 88 hours. *Sphingosine monocytogenes* grows rapidly, with a short lag phase, quickly entering the logarithmic growth phase, and a long stationary phase. In this experiment, the culture medium was used as a control, and measurements were taken under optimal wavelength conditions to minimize the influence of the culture medium on the bacterial absorbance, thus increasing the accuracy of the experiment.

[0040] like Figure 4As shown, the growth of *Sphingosphate Bacillus* is almost unaffected when the cadmium concentration is ≤12 mg / L; when the cadmium concentration is 24 mg / L, the growth of *Sphingosphate Bacillus* begins to be significantly inhibited; and *Sphingosphate Bacillus* can still grow under the condition of a cadmium concentration of 192 mg / L.

[0041] Table 1 shows the results of the analysis of variance over time for the growth characteristics of Sphingosine Bacillus under different cadmium concentrations, and Table 2 shows the results of the analysis of variance for the growth characteristics of Sphingosine Bacillus under cadmium stress at different times.

[0042] Table 1. Differences in growth characteristics of bacterial strains with different cadmium concentrations at different times.

[0043]

[0044] Table 2. Differences in bacterial growth characteristics at different time points and under different cadmium concentrations.

[0045]

[0046] Table 1 shows that when the cadmium concentration was 96 mg / L (p < 0.05), the growth characteristics of *Sphingospora* differed significantly over time; at other cadmium concentrations (p < 0.001), the growth characteristics of *Sphingospora* differed highly significantly over time, confirming the validity of the experimental results. Table 2 shows that at 0 and 8 hours (p > 0.05), the growth characteristics of *Sphingospora* at different cadmium concentrations did not differ significantly. This may be because *Sphingospora* underwent significant metabolic changes and unstable growth immediately after inoculation into the culture medium with different cadmium concentrations. At other times (p < 0.01), the growth characteristics of *Sphingospora* at different cadmium concentrations differed extremely significantly, confirming the validity of the experimental results.

[0047] Example 2: A method for regulating soil cadmium availability through the combined use of Sphingosine monophosphate and earthworms.

[0048] like Figure 6 As shown, the specific steps of this method are as follows:

[0049] S1. Select cadmium-contaminated soil, adjust the soil moisture to 60% saturation moisture content, and stabilize for 48 hours;

[0050] S2. First, add *Sphingosine monoclonal antibody* solution to the soil, then add earthworms, and incubate at 25℃ for at least 20 days; the earthworm inoculation density is 15g / kg soil; the amount of *Sphingosine monoclonal antibody* solution added is 5ml / kg soil. *Sphingosine monoclonal antibody* solution is prepared by using an inoculation loop to pick *Sphingosine monoclonal antibody* and streaking it on a *Propionibacterium* agar plate CM0827, incubating at 30℃ until a single colony grows; then, using an inoculation loop, pick a single colony and inoculate it into liquid CM0827 agar, adjusting the pH to 7.0, and incubating at a constant temperature of 30℃ for 2 days until the OD of the *Propionibacterium* agar plate CM0827 reaches a certain value.405 The value is 1.45, which is the result.

[0051] Example 3: Effects of earthworms and sphingomyelin-bacteria on cadmium availability

[0052] The test soil was tested using the method described in Example 2 above. The specific process is as follows:

[0053] The experiment set up blank soil control S, SS with only Sphingosine monoclonal bacteria, SA with only earthworms, and SSA with Sphingosine monoclonal bacteria and earthworms. (1) S: 200g soil, without adding Sphingosine monoclonal bacteria and earthworms.

[0054] (2) SS: 200g soil + 1ml Sphingomonas bacterial suspension. (3) SA: SS: 200g soil + 3g earthworms (about 5). (4) SSA: 200g soil + 3g earthworms (about 5) + 1ml Sphingomonas bacterial suspension. The Sphingomonas suspension was prepared by using an inoculation loop to pick Sphingomonas bacteria and streaking them on a Propionibacterium CM0827 agar plate, culturing at 30℃ until a single colony grew; then, using an inoculation loop, a single colony was inoculated into liquid CM0827 medium, the pH of the medium was adjusted to 7.0, and cultured at a constant temperature of 30℃ for 2 days until the OD of the Propionibacterium CM0827 medium reached a certain level. 405 The value is 1.45, which is the result.

[0055] Weigh 200g of soil that has passed through a 2mm sieve and place it in a small paper cup (75mm×53mm×90mm) with micropores at the bottom. Adjust the soil moisture to 60% saturation and allow it to stabilize for 48 hours. Inoculate earthworms into the soil where earthworms will be added; add 1ml of Sphingosine monophosphate solution to the surface of the soil where Sphingosine monophosphate will be added. To prevent earthworms from escaping, tie a mesh screen to the mouth of the cup and incubate for 20 days. Maintain a room temperature of 25℃ and keep the soil moisture content stable. After 20 days, separate the earthworms and collect the soil. After mixing the fresh soil thoroughly, quarter off a portion and store it in the refrigerator.

[0056] Cadmium in the soil was extracted by leaching with diethyltriaminepentaacetic acid (DTPA) at a ratio of 1:2.5 (Lu Rukun, 2000), and determined by atomic absorption spectrophotometer.

[0057] like Figure 5(The values ​​for each treatment in the figure are mean ± standard deviation. The experimental results were analyzed using the Turkey test, with different letters indicating statistical significance (P < 0.05); n = 3. Where: S: contaminated soil, SS: contaminated soil + Sphingosine monophosphate, SA: contaminated soil + earthworm, SSA: contaminated soil + Sphingosine monophosphate + earthworm.) As shown, the Cd-DTPA content in the soil treated with Sphingosine monophosphate was 19.92% lower than that in the original soil, while the Cd-DTPA content in the soil treated with earthworms was 3.25% higher than that in the original soil. The Cd-DTPA content in the soil treated with both Sphingosine monophosphate and earthworms was 21.54% higher than that in the original soil. These results indicate that Sphingosine monophosphate can reduce the content of available cadmium in the soil and has a significant passivation effect on cadmium; earthworms can activate cadmium in the soil and increase the bioavailability of cadmium, but this effect was not significant in this experiment (p > 0.05). This may be because the experiment only lasted 20 days, a relatively short period, and the effect of earthworms was not obvious. The content of available cadmium in the soil increased significantly under the combined action of Sphingosine mononitrate and earthworms (P<0.05).

[0058] The experimental results of this embodiment show that the method of the present invention can improve the availability of cadmium in soil. Therefore, hyperaccumulating plants (plants that absorb more than 100 times the amount of heavy metals as ordinary plants, accumulating Cr, Co, Ni, Cu, and Pb at a level generally above 110 mg / L, and Mn and Zn at a level generally above 10 mg / L (dry weight)) can be used on this soil. Examples include vetiver, centipede grass, scabra, and Indian mustard (Brassica oleracea). *Juncea* is a hyperaccumulating plant for heavy metals in soil, showing great potential for application in phytoremediation. Hyperaccumulating plants should simultaneously possess the following three basic characteristics: most of the absorbed heavy metals are distributed in the aboveground parts, i.e., a high aboveground / root concentration ratio; the concentration of a certain element within the plant exceeds a certain critical value (100 times that of ordinary plants under the same growth conditions); and it can grow normally in heavy metal-contaminated soil without exhibiting heavy metal toxicity. Examples include ferns like *Hydrangea*, wild amaranth, and the cruciferous plant *Amaranthus urinaria*, which have strong cadmium accumulation capabilities; *Solanum lyratum*, which can accumulate lead and zinc; *Artemisia* and *Mustardia*, which show significant lead accumulation; cruciferous and *Mustardia* species can be planted in nickel-contaminated soil; and *Rumex japonicus* can be planted in copper-contaminated soil, with its plants containing up to 1.850 mg / g of copper. Generally, it is necessary to consult relevant literature to accurately determine the types of hyperaccumulating plants that can absorb and remove heavy metals, thereby achieving soil remediation. This is not the focus of this example and will not be elaborated further.

[0059] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for regulating soil cadmium availability through the combined use of *Sphingomonas* and earthworms, characterized in that, Includes the following steps: S1. Select cadmium-contaminated soil, adjust the soil moisture to 60% saturation moisture content, and stabilize for 48 hours; S2. First, add *Sphingosine monoclonal antibody* solution to the soil, then add earthworms, and incubate at 25℃ for more than 20 days; the inoculation density of earthworms is 15g / kg soil; the amount of *Sphingosine monoclonal antibody* solution added is 5ml / kg soil; the preparation process of the *Sphingosine monoclonal antibody* solution is as follows: Sphingosine monoclonal antibodies were picked using an inoculation loop and streaked on a solid CM0827 Propionibacterium tumefaciens medium plate for isolation. The plates were cultured at 30°C until single colonies appeared. A single colony was then picked and inoculated into a liquid CM0827 Propionibacterium tumefaciens medium. The pH of the medium was adjusted to 7.0, and the plates were cultured at a constant temperature of 30°C for 2 days until the OD405 value of the CM0827 Propionibacterium tumefaciens medium reached 1.45, yielding the Sphingosine monoclonal antibody solution. The solid CM0827 Propionibacterium tumefaciens medium consisted of the following components: 15 g tryptone, 0.5 g L-cysteine, 5 g yeast extract, 0.5 g sodium mercaptoglycolate, 5 g glucose, 15 g agar, 2.5 g sodium chloride, 0.001 g resazurin, and 1 L distilled water. The liquid CM0827 Propionibacterium tumefaciens medium consisted of the following components: 15 g tryptone, 0.5 g L-cysteine, 5 g yeast extract, 0.5 g sodium mercaptoglycolate, 5 g glucose, 15 g agar, 2.5 g sodium chloride, 0.001 g resazurin, and 1 L distilled water. g, agar 0.7 g, sodium chloride 2.5 g, resazurin 0.001 g, distilled water 1 L; S3. Repeat steps S1-S2 once or multiple times.

2. The method for regulating soil cadmium availability by combining Sphingosine monocytogenes and earthworms according to claim 1, characterized in that: The earthworm in question is *Pleurotus sarcoptes*.

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