A river-borne Leuconostoc SCAU-D1 and its application in repairing polluted environments

Abstract

The present invention discloses a strain of Lelliottia amnigena SCAU-D1 and its application in repairing polluted environments. The present invention obtains a strain of Lelliottia amnigena SCAU-D1, which was deposited in the Guangdong Provincial Microbial Culture Collection Center on December 22, 2023, with a deposit number of GDMCC NO: 64182. The strain SCAU-D1 can effectively remove heavy metals from water bodies, including As(III), As(V), Cd, Pb, and Sb, among which As(V), Cd and Pb have excellent removal effects. Moreover, the strain SCAU-D1 can significantly reduce the effective content of heavy metals in the soil, and has high application value in the remediation and treatment of heavy metal-contaminated soil and water bodies.

Description

Technical Field

[0001] The invention belongs to the technical field of pollution remediation, and relates to a river-born Lelliottia amnigena SCAU-D1 and an application thereof in remediating a polluted environment. Background Art

[0002] Heavy metal pollution not only directly harms human health and leads to deterioration of environmental quality, but the pollution of water and soil also causes major problems in agriculture.

[0003] Existing heavy metal remediation technologies mainly include physical remediation, chemical remediation, and biological remediation, each with its own advantages and disadvantages. In particular, biological remediation includes the use of microorganisms that have the function of removing heavy metals for remediation, and plays an important role in the morphological transformation of heavy metals in soil. The use of microbial remediation is simple, economical, and environmentally friendly, and is receiving increasing attention.

[0004] Research has shown that Bacillus subtilis can absorb and utilize harmful substances such as heavy metals, converting them into harmless substances, thereby achieving environmental remediation. Therefore, Bacillus subtilis has broad application prospects in contaminated soil, wastewater treatment, and industrial and mining waste disposal.

[0005] Different microorganisms have varying effects on heavy metal remediation, and microorganisms are prone to degradation. Therefore, continuous research and expansion of the heavy metal remediation microbial library has important practical application value. Summary of the Invention

[0006] The present invention aims to explore new heavy metal remediation microorganisms. The present invention has obtained a riverine Lelliottia amnigena that can effectively remove heavy metals from water bodies and significantly reduce the effective content of heavy metals in soil. It has important application value in the remediation of heavy metal-contaminated environments such as water bodies and soil.

[0007] The purpose of the present invention is to provide a fluvial Leuconostoc SCAU-D1.

[0008] Another object of the present invention is to provide an application of the fluvial Leuconostoc SCAU-D1 in repairing heavy metal pollution.

[0009] Another object of the present invention is to provide a heavy metal pollution remediation bacterial agent containing the riverine Leyletella SCAU-D1.

[0010] Another object of the present invention is to provide a heavy metal pollution remediation method based on the fluvial Leuconostoc SCAU-D1.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention isolated a river-dwelling strain of Lelliottia amnigena, SCAU-D1, from soil in the arsenic smelting waste slag mining area of ​​Wenshan, Yunnan. Studies have shown that this strain, SCAU-D1, can effectively remove heavy metals from water and significantly reduce the available heavy metal content in soil, making it useful for remediation of heavy metal-contaminated water and soil. Therefore, the present invention claims the following:

[0013] A riverine Lelliottia amnigena SCAU-D1, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 22, 2023, with the deposit number GDMCC NO: 64182.

[0014] A bacterial agent for repairing heavy metal pollution containing the above-mentioned riverine Leuconostoc SCAU-D1.

[0015] Application of the above-mentioned Leuconostoc fluvialus SCAU-D1 or the above-mentioned bacterial agent in removing heavy metals.

[0016] Application of the above-mentioned riverine Leuconostoc SCAU-D1 or the above-mentioned bacterial agent in repairing or treating heavy metal pollution.

[0017] Application of the above-mentioned Leuconostoc fluvialus SCAU-D1 or the above-mentioned bacterial agent in repairing or treating heavy metal polluted environments.

[0018] The environment is water or soil.

[0019] The heavy metals include any one or more of As, Cd, Pb, and Sb.

[0020] Preferably, the heavy metal includes any one or more of As(V), Cd, and Pb.

[0021] A method for repairing a heavy metal-contaminated environment comprises using the above-mentioned Leuconostoc fluvialis SCAU-D1 or the above-mentioned bacterial agent for treatment.

[0022] The present invention has the following beneficial effects:

[0023] The present invention has obtained a strain of river-dwelling Lelliottia amnigena, SCAU-D1, which can effectively remove heavy metals from water, including As(III), As(V), Cd, Pb, and Sb, with particularly excellent removal of As(V), Cd, and Pb. Furthermore, SCAU-D1 can significantly reduce the available content of heavy metals in soil, making it useful for remediation of heavy metal-contaminated soil, thus possessing high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Shows the morphology and liquid culture medium of strain SCAU-D1.

[0025] Figure 2 This is the phylogenetic tree of strain SCAU-D1.

[0026] Figure 3 This is the alignment result of the 16s rDNA sequence of strain SCAU-D1 at NCBI.

[0027] Figure 4 This is the operation diagram of the aqueous solution removal test of strain SCAU-D1.

[0028] Figure 5 This is the operation diagram of the tolerance test of strain SCAU-D1.

[0029] Figure 6 Growth changes of activated culture strain SCAU-D1 under different As / Cd / Pb concentrations.

[0030] Figure 7 The growth of strain SCAU-D1 in LB medium containing different concentrations of As after 72 hours.

[0031] Figure 8 The growth of strain SCAU-D1 in LB medium containing different concentrations of Cd after 72 hours.

[0032] Figure 9 The growth of strain SCAU-D1 in LB medium containing different concentrations of Pb after 72 hours.

[0033] Figure 10 This is the operation diagram of the soil culture test of strain SCAU-D1. DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0035] Unless otherwise specified, the reagents, materials, and microorganisms used in the following examples were all commercially available.

[0036] The composition of Luria-Bertani (LB) medium: Peptone 10 g, yeast extract 5 g, sodium chloride 10 g, and water to make up to 1000 mL.

[0037] Example 1: Isolation and identification of strain SCAU-D1

[0038] 1. Sample:

[0039] The soil samples were obtained from the arsenic smelting waste slag mining area in Wenshan, Yunnan (E103°55'46"; N23°24'46").

[0040] 2. Bacteria isolation

[0041] After collecting the soil, return it to the laboratory and take 5g of sieved soil sample and place it in 95mL of sterilized LB medium. Add filter-sterilized standard arsenic (As) / standard cadmium (Cd) / standard lead (Pb) solution to make the As(Ⅲ) / As(V) concentration 50mg / L, Cd concentration 1mg / L, and Pb concentration 100mg / L. Place the sample in a constant temperature shaker under sterile conditions for incubation at 30℃ for 24h. After incubation, transfer the sample to a medium with higher concentration of As / Cd / Pb at a 1% inoculum. Cultivate under the same conditions and gradually increase the As / Cd / Pb concentration. After 5 transfers, dilute and spread the sample. Take 1mL of the supernatant and add 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 Perform gradient dilution, taking the dilution factor as 10 -5 and 10 -6 The culture medium was spread on the plate, and the colonies with good growth were streaked on the plate. The streaking was carried out three times and then stored. This was used to screen and obtain a strain with high tolerance to As / Cd / Pb, which was recorded as strain SCAU-D1.

[0042] 3. Identification of bacterial species

[0043] like Figure 1 The strain SCAU-D1 appeared white and milky in the solid culture dish, with a rough and opaque surface and irregular edges; in the liquid culture medium, the surface had a white turbidity.

[0044] The strain SCAU-D1 was sent to Beijing Aoweisen Gene Technology Co., Ltd. for 16s rDNA identification. The 16s rDNA sequence of the strain SCAU-D1 was sequenced and a developmental tree was constructed (e.g. Figure 2 shown), Figure 3 The 16s rDNA sequence of strain SCAU-D1 was aligned at NCBI and showed the highest similarity (97.76%) with the known Lelliottia amnigena strain NR 024642.1.

[0045] In summary, strain SCAU-D1 was identified as Lelliottia amnigena and was deposited in Guangdong Provincial Microbiological Culture Collection on December 22, 2023, with the deposit number GDMCC NO: 64182, and the deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0046] In the following examples, Bacillus subtilis was used as a control for conducting relevant experiments.

[0047] Example 2: Heavy metal removal experiment of strain SCAU-D1

[0048] 1. Test method

[0049] After activation, strain SCAU-D1 was inoculated into 30 mL of LB medium containing 10 mg / L As(III), 10 mg / L As(V), 1 mg / L Cd, 200 mg / L Pb, and 5 mg / L Sb, respectively. The culture was shaken at 35°C, 150 rpm, for 48 hours. The sample was then placed in a sterile centrifuge tube and centrifuged at 8000 rpm for 10 minutes. The sample was filtered through a 0.22 μm filter. 4.5 mL of the supernatant was accurately measured and placed in a digestion tube. 0.4 mL of concentrated nitric acid and 0.1 mL of concentrated hydrochloric acid were added, and microwave digestion was performed at 160°C for 10 minutes. After digestion, the solution was cooled to room temperature and transferred to a 10 mL volumetric flask. The volume was made up to 10 mL with deionized water, shaken, and then assayed. The concentration of As in the digestion solution was determined by hydride generation-atomic fluorescence spectrometry, and the concentrations of Cd and Pb in the digestion solution were determined by inductively coupled plasma optical emission spectrometry (ICP-OES).

[0050] Schematic diagram of the removal test of strain SCAU-D1 in aqueous solution Figure 4 .

[0051] 2. Test results

[0052] To evaluate the As / Cd / Pb / Sb removal efficiency of strain SCAU-D1 in aqueous solution, we measured the elemental concentrations in the supernatant of each culture medium. The results in Table 1 show that at the end of the experiment, the As(V) concentration in the supernatant decreased from 10 mg / L to 0.751 mg / L after inoculation with strain SCAU-D1, corresponding to a removal efficiency of 92.49%. While the As(III) removal efficiency was lower than that of As(V), it still showed some potential, with a removal efficiency of 26.24%. Regarding Cd and Pb, the Cd concentration in the supernatant decreased from 1 mg / L to 0.249 mg / L after inoculation with SCAU-D1, corresponding to a removal efficiency of 75.05%, and the Pb concentration in the supernatant decreased from 200 mg / L to 29.834 mg / L, corresponding to a removal efficiency of 85.08%. The Sb removal efficiency was 40.48%.

[0053] Therefore, the results showed that strain SCAU-D1 can effectively remove heavy metals in aqueous solution, including As(Ⅲ), As(Ⅴ), Cd, Pb, and Sb, among which As(Ⅴ), Cd, and Pb are particularly well removed.

[0054] Table 1. Removal effect of strain SCAU-D1 in aqueous solutions with different heavy metal (metal) concentrations

[0055]

[0056]

[0057] Example 3: Heavy metal tolerance test of strain SCAU-D1

[0058] 1. Test method

[0059] Based on the results of the aqueous solution removal test, the isolated strain SCAU-D1 was activated and cultured in the same manner as above. The strain was inoculated into 100 mL of LB medium and incubated in a shaker at 150 rpm and 30°C. After 72 hours of incubation, a 1% inoculum size (100 μL of bacterial solution) was added. SCAU-D1 was inoculated into 10 mL of culture medium containing different concentrations of As(V) (0, 0.5, 1, 2, 5, 10, 20, 30, 50, 100, 200, 300, 400 mg / L), Cd (0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 10, 20, 30 mg / L), and Pb (0, 2, 5, 10, 20, 50, 100, 200, 300, 400 mg / L). After incubation at 150 rpm and 30°C for 72 h, the OD value of the bacterial solution was determined using a multifunctional microplate reader. 600 The operation diagram of strain SCAU-D1 tolerance test is as follows Figure 5The growth changes of activated culture strain SCAU-D1 at different As / Cd / Pb concentrations are shown in Figure 6 shown.

[0060] 2. Test results

[0061] To evaluate the tolerance of Lelliottia amnigena SCAU-D1, we measured the OD value of the strain under different concentrations of As, Cd, and Pb. 600 value. Figure 7-9 The results show that:

[0062] When the initial As(V) concentration dropped below 50 mg / L, As ions showed little significant growth inhibition on SCAU-D1. Furthermore, in LB medium with As(V) concentrations of 50 mg / L or less, the strain exhibited only slight growth inhibition compared to As(V)-free medium. While growth was affected to varying degrees when the initial As(V) concentration was greater than 50 mg / L, the strain's tolerance to As(V) reached a maximum of 400 mg / L.

[0063] As for Cd, when the initial Cd concentration was lower than 1 mg / L, SCAU-D1 grew well. When it was greater than 1 mg / L, the growth of the strain was inhibited to a certain extent, but it could still maintain a high resistance, with a maximum tolerance of 30 mg / L.

[0064] When the initial Pb concentration is lower than 20 mg / L, SCAU-D1 can grow normally. In the range of 20 mg / L to 300 mg / L, although the growth of SCAU-D1 is affected to a certain extent, the maximum tolerance to Pb can reach 400 mg / L.

[0065] Therefore, the results showed that SCAU-D1 can simultaneously exhibit high tolerance to As(V), Cd, and Pb, and compared with strains resistant to single or two heavy metals, it can show excellent resistance to multiple heavy metals.

[0066] Example 4: Soil culture experiment to study the effect of strain SCAU-D1 on soil available As / Cd / Pb

[0067] 1. Test method

[0068] Based on the results of the aqueous solution removal tests, a soil experiment was conducted to investigate the effects of strain SCAU-D1 on available As / Cd / Pb in soil. Soil was collected from the surface layer (0-20 cm) of the arsenic smelting waste mine in Wenshan, Yunnan. The soil was air-dried, passed through a 2 mm sieve, and ground. Three soil types containing different heavy metal concentrations were prepared for the experiment: Soil 1 (Total As: 91.384 mg / kg), Soil 2 (Total As: 143.849 mg / kg), and Soil 3 (Total As: 270.295 mg / kg). Three soil treatments were used: untreated soil CK; soil treated with 1 mL of SCAU-D1 solution; and soil treated with 1 mL of Bacillus subtilis solution. All treatments were replicated three times, with 10 g of soil used for each treatment. The soils were watered once daily to maintain 60% of their maximum field water holding capacity. After 20 days, soil samples were collected and sieved through a 2mm sieve. Available Cd and Pb in the soil were extracted using 0.005M DTPA, and available As in the soil was extracted using 0.05M (NH4)2SO4. The As concentration in the solution was determined using hydride generation-atomic fluorescence spectrometry, and Cd and Pb concentrations were determined using ICP-OES. The experimental procedure for the soil culture of strain SCAU-D1 is shown in the figure below. Figure 10 shown.

[0069] 2. Test results

[0070] The results are shown in Tables 2 and 3.

[0071] Table 2 Effects of inoculation with SCAU-D1 and Bacillus subtilis on available As, Cd and Pb in soil 1

[0072]

[0073] Note: Different lowercase letters in the table indicate significant differences in available As / available Cd / available Pb between different treatments at the same soil concentration (p<0.05).

[0074] Table 3 Effects of inoculated strains SCAU-D1 and Bacillus subtilis on available As in soil at different concentrations

[0075]

[0076] Note: Different lowercase letters in the table indicate significant differences in available As between different treatments at the same soil concentration (p<0.05).

[0077] The immobilization capacity of the strain Lelliottia amnigena SCAU-D1 for As, Cd, and Pb was evaluated by measuring the available As, Cd, and Pb contents in contaminated soil. Table 2 shows the effect of SCAU-D1 on As, Cd, and Pb immobilization. The results show that the concentrations of all three heavy metals in the contaminated soil decreased to varying degrees. Compared with the control soil, the available As concentration in Soil 1 decreased from 0.524 mg / kg to 0.448 mg / kg under SCAU-D1 treatment, with an immobilization efficiency of 12.36%, surpassing the commonly studied effect of Bacillus subtilis. Regarding Cd, inoculation with SCAU-D1 had a significant effect, achieving an immobilization efficiency of 21%, which is comparable to the commonly studied immobilization efficiency of Bacillus subtilis. Bacteria can influence the composition of Cd in soil by affecting the binding of Cd to functional groups in the soil (such as sulfhydryls, carboxyls, and amides) and the binding of bacterial extracellular polymers to soil organic acids (Shou et al., 2018). The immobilization efficiency of SCAU-D1 on available Pb was comparable to that of Bacillus subtilis, with both achieving an immobilization rate of 20%. The results showed that SCAU-D1 significantly reduced the concentrations of available As, Cd, and Pb in contaminated soils, and exhibited a higher As immobilization rate than Bacillus subtilis.

[0078] The immobilization capacity of SCAU-D1 was evaluated by measuring the available As content in different contaminated soils. The results in Table 3 show that the available As concentrations in different soils decreased significantly after SCAU-D1 treatment. As the As concentration increased, SCAU-D1 was more effective than Bacillus subtilis in immobilizing As. In the high-concentration As-contaminated soils Soil 2 and Soil 3, the immobilization effect of SCAU-D1 in Soil 3 was significantly better than that in Soil 2.

[0079] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A strain of Leuconostoc fluvialensis ( Lelliottia amnigena ) SCAU-D1, characterized by, The bacterium was deposited in Guangdong Provincial Microbiological Culture Collection Center on December 22, 2023, with the deposit number GDMCC NO: 64182.

2. A bacterial agent for repairing heavy metal pollution, characterized in that: Contains the fluvial Leuconostoc SCAU-D1 according to claim 1, wherein the heavy metal is any one or more of As (III), As (V), Cd, Pb, and Sb.

3. Use of the fluvial L. SCAU-D1 according to claim 1 or the bacterial agent according to claim 2 for removing heavy metals, wherein the heavy metals are any one or more of As (III), As (V), Cd, Pb, and Sb.

4. Use of the fluvial Leuconostoc SCAU-D1 according to claim 1 or the bacterial agent according to claim 2 in the repair or treatment of heavy metal pollution, wherein the heavy metal is any one or more of As (III), As (V), Cd, Pb, and Sb.

5. Use of the fluvial Leuconostoc SCAU-D1 according to claim 1 or the bacterial agent according to claim 2 in repairing or treating a heavy metal-contaminated environment, wherein the heavy metal is any one or more of As(III), As(V), Cd, Pb, and Sb.

6. The application according to claim 5, characterized in that: The environment is soil or water.

7. A method for repairing a heavy metal polluted environment, characterized in that: The treatment is carried out using the fluvial Leuconostoc SCAU-D1 described in claim 1 or the bacterial agent described in claim 2, and the heavy metal is any one or more of As (III), As (V), Cd, Pb, and Sb.

8. The method according to claim 7, characterized in that: The environment is soil or water.

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