Cd-resistant bacillus cereus as well as application and method of Cd-resistant bacillus cereus in remediation of Cd-polluted soil

Abstract

The invention relates to the technical field of phytoremediation of Cd-polluted soil. The invention provides Cd-resistant bacillus cereus as well as application and a method of the Cd-resistant bacillus cereus in remediation of Cd-polluted soil, the preservation name of the bacillus cereus is Bacillus cereus 01, the Latin name of the bacillus cereus is Bacillus cereus, the bacillus cereus is preserved in Guangdong Microbial Culture Collection Center, the preservation number of the bacillus cereus is GDMCC No: 65570, and the preservation time is December 2, 2024. The efficient Cd-resistant endophytic bacteria are obtained through screening and identification, the Cd-resistant endophytic bacteria are combined with the smooth vetch to repair Cd-polluted soil, absorption of succeeding crops to Cd is reduced, the food safety problem caused by standard exceeding of Cd is reduced, and the method has the advantages of being low in cost, high in repair efficiency, simple and convenient in treatment step and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of phytoremediation of Cd-polluted soil, and particularly relates to a Bacillus cereus resistant to Cd and its application and method in repairing Cd-polluted soil. Background Art

[0002] The background content of Cd in global soil is 0.01 - 2.00 mg / kg, and the median is 0.35 mg / kg. Soil heavy metal pollution first affects its own environment, and changes occur in soil physical and chemical properties, microbial communities, and enzyme activities. Under the stress of heavy metal Cd, the soil pH can be reduced, the bulk density and cation exchange capacity can be decreased, the organic matter in the soil can be degraded, and it can also cause deficiencies in available nutrients such as available potassium, alkaline hydrolyzable nitrogen, and available phosphorus and the reduction of their effectiveness, resulting in the weakening of the soil's ability to supply nutrients to crops; for plants, when the Cd in the soil exceeds a certain concentration, it has a greater impact on plant growth, and may inhibit plant growth and reduce the above-ground biomass. Cd stress can also lead to the production of reactive oxygen species (ROS) at the subcellular level in plants, causing damage to cell membranes and biomolecules and organelles. Excessive Cd reduces the photosynthetic pigments and gas exchange characteristics of plants, inhibiting plant photosynthesis. Therefore, it is urgent to repair the Cd pollution in local farmland soil.

[0003] How to efficiently treat the combined pollution of soil uranium and Cd is a major problem faced by mankind. Soil heavy metal pollution remediation technologies include physical and chemical remediation and agricultural remediation methods. Compared with traditional physical and chemical remediation technologies, phytoremediation technology has the advantages of environmental friendliness, non-destruction of the soil ecological environment, low cost, and aesthetics. Therefore, the phytoremediation technology for treating soil Cd combined pollution has been favored by many researchers in the environmental field and even the agricultural field. Vicia villosa var. is an annual leguminous plant discovered in recent years that can improve soil fertility, reduce the application amount of chemical fertilizers, and enhance the resistance of crops, and plays an irreplaceable role in ensuring high crop yields and constructing a good agricultural ecological environment. Vicia villosa var. contains many endophytes with multiple functions, among which there are endophytes with the ability to passivate heavy metals, and existing research has shown that planting Vicia villosa var. can increase the abundance of microorganisms in the soil, passivate soil Cd, and improve crop yields and resistance. Therefore, using Vicia villosa var. to treat soil Cd pollution has great advantages.

[0004] However, the environmental effects of heavy metal-contaminated soil are often complex and variable due to various external factors. There may be multiple heavy metals, which can hinder the growth of hyperaccumulator plants and greatly reduce their ability to remediate heavy metals. Therefore, while ensuring the growth of hyperaccumulator plants, enhancing their efficiency in remediating Cd pollution has become the key to soil heavy metal remediation. Cd-tolerant bacteria have stronger adaptability to Cd-contaminated soil. Cd-tolerant bacteria can reduce the toxicity of Cd to plants by complexing Cd in the soil, and can also promote plant growth through nitrogen fixation, phosphorus solubilization, production of siderophores and auxin. In addition, Cd-tolerant bacteria contain extracellular polymeric substances (EPS), which are substances secreted by microorganisms to adapt to changes in external conditions. Cd stress will stimulate the production of EPS. There are a large number of anionic groups (carboxyl, hydroxyl, amino, etc.) in EPS, which can adsorb or chelate with metal ions, thus effectively removing metal or heavy metal ions contained in the soil solution. In addition, the combined use of plants and microorganisms in phytoremediation has a unique role in remediating heavy metal-contaminated soil. Microorganisms themselves can adsorb heavy metals. At the same time, microorganisms interact with the roots and root exudates of hyperaccumulator plants, changing the rhizosphere microenvironment, reducing the absorption of heavy metals in the soil by the roots and the transfer of Cd to the above-ground parts, thereby reducing the Cd content in agricultural products, or promoting the absorption of plant roots, increasing the transport of Cd to the above-ground parts, and then achieving the removal of Cd in the soil and reducing the Cd content in the soil by harvesting the above-ground parts of the plants.

[0005] At present, the combined use of microorganisms and plants in remediating heavy metal pollution is widely applied. However, the use of the endophytic bacterium Bacillus cereus and the leguminous plant Vicia villosa Roth to remediate Cd-contaminated soil has not been reported yet, and endophytic bacteria can colonize inside plants. Therefore, screening Cd-tolerant endophytic bacteria can not only enrich the microbial remediation strain library, but also apply the screened Cd-tolerant endophytic bacteria and phytoremediation together to the treatment of Cd-contaminated soil, which can not only effectively reduce the bioavailability of Cd in the soil, but also increase the systemic resistance of the subsequent crops while inhibiting the absorption of Cd by the plant roots, and has important practical significance for the treatment of Cd-contaminated soil. Summary of the Invention

[0006] The purpose of the present invention is to provide a Cd-tolerant Bacillus cereus and its application and method in remediating Cd-contaminated soil, so as to reduce the absorption of Cd by subsequent crops and reduce food safety problems caused by excessive Cd.

[0007] In order to achieve the above invention purpose, the present invention provides the following technical solutions:

[0008] The present invention provides a Cd-tolerant Bacillus cereus. The preservation name of the Bacillus cereus is Bacillus cereus s 01, and the Latin name is Bacillus cereus. It is preserved in the Guangdong Microbial Culture Collection Center, with the preservation number GDMCC No: 65570, and the preservation date is December 2, 2024.

[0009] The present invention also provides the application of the above-mentioned Bacillus cereus in repairing Cd-polluted soil.

[0010] The present invention also provides a method for repairing Cd-polluted soil using the above-mentioned Bacillus cereus, which includes the following steps:

[0011] (1) Disinfect the seeds of Vicia villosa, soak them, and then sow them.

[0012] (2) After the emergence of Vicia villosa, irrigate the bacterial suspension of the above-mentioned Bacillus cereus.

[0013] (3) When Vicia villosa grows to the full-bloom stage, harvest it, and then return the plants to the field.

[0014] Preferably, in step (1), the disinfection method is: soak the seeds of Vicia villosa in a 0.8-1.2% sodium hypochlorite solution for 25-35 min, then rinse them with water 3-5 times. The soaking time is 10-14 h, the sowing depth is 0.5-1.2 cm, and water is poured once every 2-4 d after sowing.

[0015] Preferably, in step (2), the irrigation period is when the seedlings of Vicia villosa grow to 2.5-3.5 cm, the number of irrigation times is 2-4 times, and the irrigation interval is 8-12 d.

[0016] Preferably, the preparation method of the bacterial suspension in step (2) includes the following steps:

[0017] A. Inoculate the above-mentioned Bacillus cereus into an LB solid medium and culture it at 28-32 °C for 20-28 h to obtain an activated Bacillus cereus.

[0018] B. Inoculate the activated Bacillus cereus into an LB liquid medium and shake-culture it at 28-32 °C and 180-220 rpm for 60-84 h to obtain a fermentation broth.

[0019] C. Centrifuge the fermentation broth at 2-6 °C for 10-20 min to obtain a cell precipitate.

[0020] D. Configure the cell precipitate with water into a bacterial suspension of 0.8-1.2×10 8 CFU / mL.

[0021] The present invention provides a cadmium-resistant Bacillus cereus and its application and method for repairing cadmium-polluted soil. The preservation name of the Bacillus cereus is Bacillus cereuss 01, the Latin name is Bacillus cereus, it is preserved in the Guangdong Provincial Culture Collection of Microorganisms, the preservation number is GDMCC No: 65570, and the preservation time is December 2, 2024. Through screening and identification, the present invention obtains an endophytic bacterium with high cadmium resistance. Using this cadmium-resistant endophytic bacterium in combination with Vicia villosa to repair cadmium-polluted soil can reduce the absorption of cadmium by subsequent crops and reduce food safety problems caused by excessive cadmium. This method has the advantages of low cost, high repair efficiency, and simple treatment steps.

[0022] Preservation description

[0023] A cadmium-resistant Bacillus cereus, the preservation name of the Bacillus cereus is Bacillus cereuss01, the Latin name is Bacillus cereus, it is preserved in the Guangdong Provincial Culture Collection of Microorganisms, the preservation number is GDMCCNo: 65570, the preservation time is December 2, 2024, and the preservation address is the 5th floor of Building 59, No. 100 compound, Middle Xianlie Road, Guangzhou. Description of the drawings

[0024] Figure 1 It is the process flow of the method for repairing cadmium-polluted soil by combining Bacillus cereus and Vicia villosa.

[0025] Figure 2 It is the growth morphology and Gram staining of Bacillus cereus.

[0026] Figure 3 It is the phylogenetic tree of Bacillus cereus.

[0027] Figure 4 It is the influence of environmental factors on the removal of cadmium by Bacillus cereus 2+ (where A is the pH value; B is the temperature; C is the rotation speed; D is the inoculum size). According to the analysis of variance and Tukey test (P<0.05), different letters (a, b, c, d) indicate significant differences between different treatments within the same group; the error bars represent mean ± standard deviation.

[0028] Figure 5 It is the influence of environmental factors on the adsorption of cadmium by Bacillus cereus 2+ (where A is the pH value; B is the temperature; C is the rotation speed; D is the inoculum size). According to the analysis of variance and Tukey test (P<0.05), different letters (a, b, c, d) indicate significant differences between different treatments within the same group; the error bars represent mean ± standard deviation.

[0029] Figure 6 Bacillus cereus CdG01 in the presence of different concentrations of Cd 2+ Changes in pH value (A) and growth amount (B) in LB medium with (25, 50, 75, 100, 125 mg / L).

[0030] Figure 7 For different Cd 2+ The effect of CdG01 on Cd in LB medium 2+ Removal rates of 25 mg / L, 50 mg / L, 75 mg / L, 100 mg / L and 125 mg / L, according to analysis of variance and Tukey test (P<0.05), different letters indicate significant differences among different treatments in the same group; error bars represent mean ± SD.

[0031] Figure 8 Effects of different treatments on the growth and Cd accumulation characteristics of V. glossyleaf. Among them, A, C, and E on the left show the actual growth conditions of V. glossyleaf only; B, D, and E on the right show the actual growth conditions of V. glossyleaf + watering with CdG01 bacterial suspension; F is plant height; G is root length; H is the fresh weight of the aboveground part; I is the fresh weight of the underground part; J is the change in the number of nodules; K is the effect of different treatments on the Cd content in the aboveground and underground parts of V. glossyleaf. According to analysis of variance and Tukey's test (P<0.05), different letters indicate significant differences among different treatments in the same group; error bars represent mean ± standard deviation.

[0032] Figure 9 Figure 2 Effects of Vetch on available Cd content in soil before and after turning over. Different letters indicate significant differences among treatments in the same group according to analysis of variance and Tukey test (P<0.05); error bars represent mean ± SD.

[0033] Figure 10 The figures show the effects of turning over and before of glossy-leaved vetch on the growth of cabbage, where A is the blank control; B is only watering with CdG01 bacterial suspension; C is only turning over glossy-leaved vetch; D is turning over glossy-leaved vetch + watering with CdG01 bacterial suspension; E is yield; F is leaf length; G is leaf width; H is the number of leaves; I is the dry weight of the aboveground part; J is the dry weight of the underground part. According to analysis of variance and Tukey's test (P<0.05), different letters indicate significant differences among different treatments in the same group; error bars represent mean ± standard deviation.

[0034] Figure 11Effects of Vicia villosa before and after incorporation on Cd accumulation in Chinese cabbage. Among them, A represents the Cd content in the soil under different treatments; B represents the Cd content in Chinese cabbage under different treatments; according to the analysis of variance and Tukey test (P<0.05), different letters indicate significant differences between different treatments within the same group; error bars represent mean ± standard deviation. Specific implementation manners

[0035] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0036] Example 1 Screening of Cd-tolerant endophytic bacteria in the roots of Vicia villosa

[0037] 1. Collection of Vicia villosa samples

[0038] The collected Vicia villosa was Vicia villosa var. growing in a moderately Cd-polluted farmland in the south, which has certain tolerance and accumulation ability to Cd. The collection method was to randomly dig up healthy Vicia villosa plants with roots and soil. After the samples were put into pre-prepared clean fresh-keeping bags together with rhizosphere soil, they were taken back to the laboratory and stored at 4°C for the isolation of root endophytic bacteria;

[0039] 2. Isolation of Cd-tolerant endophytic bacteria

[0040] The collected roots of Vicia villosa were rinsed clean with sterile water, air-dried indoors, and then, under sterile conditions, 10 g of the washed plant roots were weighed, surface-sterilized with 75% ethanol, cut into 3-cm segments with a sterile scalpel, soaked in 75% ethanol for 1 min, and then rinsed twice with sterile water. Then, soaked in 0.1% mercuric chloride for 30 s, rinsed three times with sterile water, put into a sterilized mortar containing 9 mL of sterile water, added a little sterilized quartz sand and ground evenly, and left to stand for 15 min. Take 1 mL and dilute it to 10 -4 、10 -5 、10 -6 concentration gradients. Take 0.1 mL from each concentration gradient suspension and spread it on an LB solid medium with a Cd 2+ concentration of 100 mg / L (CdCl 2100mg, agar 15g / L, yeast powder 5.0g / L, NaCl 10.0g / L, tryptone 10.0g / L, dilute to 1000mL with distilled water, adjust pH to 7.0, sterilize at 121℃ for 20min) on a plate and invert at 30℃, with the sterile water used to wash the sample for the last time as a control. Observe for 7 consecutive days. When colonies grow on the surface of the solid culture medium, pick single colonies of different shapes and sizes and inoculate them on a new LB solid culture medium (agar 15g / L, 15 yeast powder 5.0g / L, NaCl 10.0g / L, tryptone 10.0g / L, dilute to 1000mL with distilled water, adjust pH to 7.0, and sterilize at 121℃ for 20min) for streak culture to isolate single-morphological strains. When colonies grow on the new solid culture medium, purify twice more to isolate multiple single strains. The results showed that one strain could tolerate 100 mg / L Cd. 2+ The strain with a concentration of 1.577 mmol / l was named CdG01 and deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No: 65570. The deposit date is December 2, 2024. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Temporary preservation of strains: The Cd-resistant strains were inoculated with colonies picked up with an inoculation needle and inoculated onto NA solid slant medium and stored in a refrigerator at 4°C. For cold storage of strains, the purified strains must be stored in sterile glycerol at -80°C.

[0041] Example 2 Morphological characteristics, physiological and biochemical and molecular biological identification of strain CdG01

[0042] The colonies formed by the strain CdG01 isolated by the above method on the surface of LB solid medium are characterized by round pale yellow-white colonies, dry and sticky surface, ridges, wrinkles in the middle, regular wavy edges, and a pungent odor ( Figure 2 A and B show two colony morphologies of strain CdG01. Under an electron microscope, the bacteria are short rod-like structures with wrinkled and uneven surfaces. ( Figure 2 C shows the electron microscopic structure of strain CdG01).

[0043] To further study the physiological and biochemical activities of CdG01, the physiological and biochemical determination of CdG01 was carried out using the Biolog method. The physiological and biochemical determination of bacteria was performed using the Biolog microplate method according to the Biolog GenⅢ microplate operation manual. The specific operation was to inoculate the pure cultured bacterial strain into the BUG agar medium and culture it at 33 °C; then use an Inoculatorz cotton swab to pick up the colonies from the plate, and then insert the end of the cotton swab into the bottom of the inoculation tube containing the inoculation liquid, shake it up and down to release the bacteria into the inoculation liquid, and stir evenly to obtain a cell suspension, which was measured with a turbidimeter and adjusted to the required concentration; then the bacterial suspension was added to the identification plate and the lid was covered, and after 24 h and 48 h respectively, it was placed in the Biolog reader for measurement. The results showed that: in terms of physical and chemical properties, CdG01 was positive at pH values of 5-10, the growth temperature was 15-45 °C, and the bacterium was Gram-positive stained ( Figure 2 D), and was also positive under 0-8% NaCl. In addition, CdG01 could hydrolyze milk, starch, Tween 20, Tween 80, and cellulose, and was also positive in nitrate reduction, arginine dihydrolase, esculin hydrolysis, and gelatin hydrolysis, that is, CdG01 could grow normally under these conditions. In terms of carbon source utilization, CdG01 could decompose and utilize carbon sources such as D-glucose, N-acetylglucosamine, D-maltose, malic acid, trisodium citrate, etc., while it was negative for L-arabinose, D-mannose, D-dextromannose, potassium gluconate, caprylic acid, adipic acid, phenylacetic acid, etc., that is, it could not be fully utilized. In addition, the ZYM reagent conditions of CdG01 were tested, and CdG01 was positive for alkaline phosphatase, esterase (C4), lipase (C8), leucine arylamidase, and α-glucosidase, and negative for lipase (C14), valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, β-glucosidase, N-acetyl-glucosaminidase, α-mannosidase, and β-fucosidase (Table 1). Generally speaking, CdG01 could adapt to a variety of environments and could decompose and utilize a variety of organic substances, which was in line with the physiological and biochemical characteristics of Bacillus.

[0044] Table 1 Biolog identification results of CdG01

[0045]

[0046]

[0047] Note: "+" indicates positive, and "-" indicates negative

[0048] The purified strain (CdG01) was identified by 16S rRNA amplification and sequence analysis. The specific method was as follows: The genomic DNA of the purified strain was extracted using the TSINGKE Plant DNA Extraction Kit (universal type). The 16S rDNA of this strain was amplified using the following universal primers (synthesized by Beijing Tsingke Biotechnology Co., Ltd.): 27F (upstream primer, sequence: 5'-agtttgatcmtggctcag-3', SEQ ID NO.1) and 1492R (downstream primer, sequence: 5'-ggttaccttgttacgactt-3', SEQ ID NO.2). The amplification system and conditions are shown in Tables 1 and 2 below. The amplification product was sent to Beijing Tsingke Biotechnology Co., Ltd. for sequencing.

[0049] Table 2 16S rRNA Amplification System

[0050]

[0051]

[0052] Table 3 16S rRNA Amplification Conditions

[0053]

[0054] The PCR product obtained by amplification through the above method was uploaded to the NCBI database with its sequencing results, and BLAST alignment was performed on the website. Using the MEGA 11 software, a phylogenetic tree was constructed using the Neighbor-Joining method to determine the taxonomic status of the isolated endophytic strain CdG01.

[0055] The assembled sequence of strain CdG01 is shown as SEQ ID NO.3:

[0056] SEQ ID NO.3:

[0057]

[0058] According to the classification results of the phylogenetic tree (see Figure 3 ), the strain CdG01 has the closest genetic relationship with Bacillus cereus XaM6 and clusters into one branch with a sequence similarity of 100%. Based on the culture characteristics, morphological characteristics, physiological and biochemical characteristics of the strain CdG01 and the analysis results of the 16S rRNA sequence, this strain is determined to be Bacillus cereus CdG01.

[0059] Example 3 Evaluation of the Optimal Culture Conditions for Cd Adsorption by CdG01 2+

[0060] Although there are many studies on the adsorption of heavy metal ions by bacterial cells, the adsorption process is intricate. In view of this, this example studied the characteristics of Cd adsorption by CdG01 cells, analyzed the effects of different initial pH values, culture temperatures, inoculum amounts, and different culture rotation speeds on the Cd 2+ adsorption effect of cells to determine the optimal adsorption conditions. 2+

[0061] Dissolve CdCl 2 in sterile water to prepare a mother liquor with a concentration of 1000 mg / L of Cd 2+ , and dilute the mother liquor to the required concentration according to the subsequent test conditions. Prepare the freeze-dried powder of CdG01 cells. Take out the frozen CdG01 strain from the -80 °C refrigerator, inoculate it into LB broth medium, and perform resuscitation culture at 30 °C and 200 rpm until the bacterial solution reaches the logarithmic growth phase. When the bacterial solution reaches an appropriate growth density (OD 600 value reaches 0.8), aliquot it into 50 mL sterile centrifuge tubes, centrifuge at 6000 rpm and 4 °C for 15 min to collect the cells, wash the centrifuged cell precipitate with sterile normal saline to remove the medium residues, and repeat the washing 3 times. After washing the cells well, put a sterile breathable sealing film on the mouth of the 50 mL centrifuge tube, place it in a -20 °C refrigerator for pre-freezing to completely freeze the sample, put the pre-frozen sample into the freeze-drying chamber of the freeze-dryer, and freeze-dry at -80 °C for 12 h. After freeze-drying, aliquot the freeze-dried cells into sterile freeze-drying tubes and store them in a -20 °C refrigerator for standby.

[0062] Prepare 50 mL of LB medium with a Cd 2+ concentration of 100 mg / L, adjust the pH value to 4.0, 5.0, 6.0, 7.0, and 8.0 with 1 M HCl or NaOH, and add 0.05 g of freeze-dried cells to the above Cd 2+ ​​In an LB medium with a concentration of 100 mg / L, oscillate and react at 30 °C and 180 rpm for 48 h to explore the effect of different pH values on the adsorption of Cd by CdG01 2+ Similarly, set up adsorption systems with different temperatures at pH 7, and the culture temperatures are 22, 25, 28, 30, and 33 °C respectively. Put 0.05 g of freeze-dried bacterial cells and oscillate and react at 180 rpm for 48 h to explore the effect of different culture temperatures on the adsorption of Cd by CdG01 2+ Set up adsorption systems with different culture rotation speeds at pH 7, the culture temperature is 30 °C, put 0.05 g of freeze-dried bacterial cells, and oscillate and react at 130, 150, 180, 200, and 220 rpm for 48 h to explore the effect of different culture rotation speeds on the adsorption of Cd by CdG01 2+ Set up adsorption systems with different inoculum amounts at pH 7, the culture temperature is 30 °C, and the amounts of freeze-dried bacterial cells put in are 0.02, 0.05, 0.08, 0.10, and 0.15 g respectively. Oscillate and react at 180 rpm for 48 h to explore the effect of different inoculum amounts on the adsorption of Cd by CdG01 2+ After the above biological adsorption process is completed, centrifuge at 10000 rpm for 15 min, and use ICP-MS to measure the Cd concentration in the supernatant 2+ The experiment is set with 3 repeated treatments, and a blank control is set at the same time. Use formulas (1) and (2) to calculate the Cd removal rate and adsorption capacity 2+ The optimal culture conditions for the adsorption of Cd by CdG01 were evaluated, and the results are as shown in

[0063]

[0064] C 0 and Ce refer to the initial and equilibrium metal ion concentrations respectively; m (g) is the dry weight of the microbial biosorbent, and V (L) is the volume of the working solution

[0065] For the adsorption of Cd by CdG01 2+ The best culture conditions were evaluated, and the results are as shown in Figure 4 and 5 shown. Under different culture conditions, the target strain showed different Cd removal effects. When the pH value of the adsorption system was set to 5, CdG01 had the highest Cd removal rate and adsorption capacity( 2+ A, 2+ A). Under different culture temperature conditions, CdG01 showed higher Cd removal rate and adsorption capacity at 30 °C, and was significantly higher than other temperature treatments (P < 0.05)( Figure 4 A, Figure 5 A). In addition, when the rotation speed was 200 rpm, CdG01 had the highest Cd removal rate and adsorption capacity for Cd 2+ removal rate and adsorption capacity, and was significantly higher than other rotation speed treatments (P < 0.05)( Figure 4 B, Figure 5 B). In addition, when the rotation speed was 200 rpm, CdG01 had the highest Cd removal rate and adsorption capacity for Cd 2+The effects on the removal rate and adsorption capacity were more obvious (P<0.05) and reached the maximum value ( Figure 4 C, Figure 5 C). When the inoculum size was 0.08 g, the Cd removal rate by CdG01 was the highest, and there were significant differences compared with other groups (P<0.05)( 2+ D). In addition, when the inoculum size was 0.02 g, the adsorption capacity of CdG01 for Cd Figure 4 was the highest, and there were significant differences compared with other groups (P<0.05, 2+ D). This difference was attributed to the different effects of the inoculum size on the adsorption capacity, resulting in the differences. Since the Cd removal rate by bacteria was relatively low when the inoculum size was 0.02 g, the adsorption capacity and adsorption efficiency of bacteria must be considered simultaneously in the actual adsorption scheme. Therefore, considering comprehensively, an inoculum size of 0.08 g was selected as the optimal inoculum size for CdG01. In summary, the optimal culture conditions for CdG01 to adsorb Cd Figure 5 were a pH value of 5, a culture temperature of 30 °C, a rotation speed of 200 rpm, and an inoculum size of 0.08 g. 2+ 2+ 2+ 2+

[0066] Example 4 Effects of different initial cadmium concentrations on the adsorption of Cd 2+ by the strain

[0067] Using 250 mL conical flasks as containers, the effects of different initial concentrations of Cd 2+ on the growth state of CdG01 and Cd adsorption were studied. CdCl 2 was used to prepare a mother liquor with a mass concentration of 1000 mg / L Cd 2+ and added to the LB liquid medium to make the Cd 2+ concentration in the LB liquid medium 25, 50, 75, 100, and 125 mg / L. The LB liquid medium without adding the Cd 2+ mother liquor was used as a blank control. After autoclaving at 121 °C for 30 min, the adsorption system was set according to the optimal culture conditions for CdG01 to adsorb Cd 2+ determined in Example 3, with a pH value of 7, a culture temperature of 30 °C, an input amount of freeze-dried bacteria of 0.08 g, and a rotation speed of 200 rpm. The samples were taken at 6, 12, 24, 48, 72, 120, 144, and 192 h after continuous culture in a constant temperature shaking incubator. After centrifuging at 10000 rpm for 15 min, the Cd 2+ concentration in the supernatant was measured by ICP-MS. The experiment was set with 3 repeated treatments and a blank control, and the Cd 2+ concentration was calculated using formulas (1) and (2) in Example 3. 2+Removal rate; Measure the OD of the culture solution with a visible-ultraviolet spectrophotometer (zero with deionized water), and measure the pH value with a pH meter. 600 Value (zero with deionized water), and measure the pH value with a pH meter.

[0068] Figure 6 Figure 6 shows the changes in pH value and growth amount (OD 600 ) of Bacillus cereus CdG01 in LB medium with different initial cadmium concentrations (0, 25, 50, 75, 100, 125 mg / L). Figure 6 A shows the trend of pH value changing with different initial cadmium concentrations at different times. At a cadmium concentration of 25 mg / L, the pH value slightly decreased at the initial stage of cultivation and then gradually stabilized, with a pH value of 8.88 at 192 h; at a cadmium concentration of 50 mg / L, the pH value decreased more significantly at the initial stage of cultivation and then gradually stabilized, with a pH value of 8.96 at 192 h; at a cadmium concentration of 75 mg / L, the pH value decreased even more significantly at the initial stage of cultivation, with a pH value of 8.85 at 192 h; at a cadmium concentration of 100 mg / L, the pH value decreased significantly at the initial stage of cultivation, with a pH value of 8.75 at 192 h; at a cadmium concentration of 125 mg / L, the pH value decreased most significantly at the initial stage of cultivation, with a pH value of 8.53 at 192 h. On the other hand, 2+ Concentration, the pH value slightly decreased at the initial stage of cultivation and then gradually stabilized, with a pH value of 8.88 at 192 h; at a cadmium concentration of 50 mg / L, the pH value decreased more significantly at the initial stage of cultivation and then gradually stabilized, with a pH value of 8.96 at 192 h; at a cadmium concentration of 75 mg / L, the pH value decreased even more significantly at the initial stage of cultivation, with a pH value of 8.85 at 192 h; at a cadmium concentration of 100 mg / L, the pH value decreased significantly at the initial stage of cultivation, with a pH value of 8.75 at 192 h; at a cadmium concentration of 125 mg / L, the pH value decreased most significantly at the initial stage of cultivation, with a pH value of 8.53 at 192 h. On the other hand, 2+ Concentration, the pH value decreased more significantly at the initial stage of cultivation and then gradually stabilized, with a pH value of 8.96 at 192 h; at a cadmium concentration of 75 mg / L, the pH value decreased even more significantly at the initial stage of cultivation, with a pH value of 8.85 at 192 h; at a cadmium concentration of 100 mg / L, the pH value decreased significantly at the initial stage of cultivation, with a pH value of 8.75 at 192 h; at a cadmium concentration of 125 mg / L, the pH value decreased most significantly at the initial stage of cultivation, with a pH value of 8.53 at 192 h. On the other hand, 2+ Concentration, the pH value decreased even more significantly at the initial stage of cultivation, with a pH value of 8.85 at 192 h; at a cadmium concentration of 100 mg / L, the pH value decreased significantly at the initial stage of cultivation, with a pH value of 8.75 at 192 h; at a cadmium concentration of 125 mg / L, the pH value decreased most significantly at the initial stage of cultivation, with a pH value of 8.53 at 192 h. On the other hand, 2+ Concentration, the pH value decreased significantly at the initial stage of cultivation, with a pH value of 8.75 at 192 h; at a cadmium concentration of 125 mg / L, the pH value decreased most significantly at the initial stage of cultivation, with a pH value of 8.53 at 192 h. On the other hand, 2+ Concentration, the pH value decreased most significantly at the initial stage of cultivation, with a pH value of 8.53 at 192 h. On the other hand, Figure 6 B shows the trend of the growth amount of the strain changing with different initial cadmium concentrations at different times. At a cadmium concentration of 25 mg / L, the growth amount of the strain also gradually increased within 192 h, but the growth rate was slow, and the OD 2+ Concentration, the growth amount of the strain gradually increased within 192 h, but the growth rate was slow, and the OD 600 Value was 2.24 at 192 h; at a cadmium concentration of 50 mg / L, the growth amount of the strain increased within 192 h, but the growth rate further slowed down, and the OD 2+ Concentration, the growth amount of the strain increased within 192 h, but the growth rate further slowed down, and the OD 600 Value was 2.50 at 192 h; at a cadmium concentration of 75 mg / L, the growth amount of the strain increased within 192 h, but the growth rate significantly slowed down, and the OD 2+ Concentration, the growth amount of the strain increased within 192 h, but the growth rate significantly slowed down, and the OD 600 Value was 2.39 at 192 h; at a cadmium concentration of 100 mg / L, the growth amount of the strain increased within 192 h, but the growth rate significantly slowed down, and the OD 2+ Concentration, the growth amount of the strain increased within 192 h, but the growth rate significantly slowed down, and the OD 600 Value was 2.39 at 192 h; at a cadmium concentration of 125 mg / L, the growth amount of the strain increased within 192 h, but the growth rate was the slowest, and the OD 2+ Concentration, the growth amount of the strain increased within 192 h, but the growth rate was the slowest, and the OD 600 Value was 1.97 at 192 h.

[0069] In summary, from the perspective of pH value changes, as the initial cadmium concentration increases, the pH value of the culture medium decreases at the initial stage of cultivation, but stabilizes at 192 h. Specifically, at Cd concentrations of 25 mg / L and 50 mg / L, the pH values are 8.88 and 8.96 at 192 h, respectively, indicating that at lower concentrations, the pH value changes less and tends to be stable. At higher concentrations (75 mg / L, 100 mg / L, and 125 mg / L), the pH values are 8.85, 8.75, and 8.53 at 192 h, respectively, indicating that as the cadmium concentration increases, the pH value decreases more significantly. From the perspective of growth quantity changes, the growth quantity (OD 600 ) of the strain increases with time at all concentrations, but the growth rate slows down at higher concentrations. At Cd concentrations of 25 mg / L and 50 mg / L, the OD 600 values at 192 h are 2.24 and 2.50, respectively, indicating that at lower concentrations, the growth quantity of the strain is higher and the growth rate is faster. At higher concentrations (75 mg / L, 100 mg / L, and 125 mg / L), the OD 600 values at 192 h are 2.39, 2.39, and 1.97, respectively, indicating that as the cadmium concentration increases, the growth quantity of the strain decreases and the growth rate slows down significantly, indicating that high-concentration cadmium has a certain inhibitory effect on the growth of the strain. Through comprehensive analysis, Bacillus cereus CdG01 shows good adaptability and growth ability in cadmium environments at lower concentrations (25 mg / L and 50 mg / L), with less pH value change and higher growth quantity. At higher concentrations (75 mg / L, 100 mg / L, and 125 mg / L), although the strain can still grow, the pH value decreases more significantly, the growth quantity decreases, and the growth rate slows down, indicating that high-concentration cadmium has a certain inhibitory effect on the growth of the strain. These results indicate that Bacillus cereus CdG01 has good remediation potential in cadmium-polluted environments at lower concentrations and can be effectively applied to the remediation of cadmium-polluted soil.

[0070] In Example 4 of the present invention, the effects of different initial Cd 2+ concentrations (25, 50, 75, 100, 125 mg / L) on the adsorption performance of the endophytic bacterium Bacillus cereus CdG01 were studied, and the experimental results are as Figure 7 shown. Specifically, Figure 7 it is divided into five parts (A - E), corresponding to different initial Cd2+ concentrations (25, 50, 75, 100, and 125 mg / L) respectively. Each part shows the removal rate of CdG01 for Cd 2+ at different time points (6, 12, 24, 48, 72, 120, 144, 192 h), and the significant differences between time points are marked by analysis of variance and Tukey test (P < 0.05). Initial Cd2+ Analysis of the effect of concentration on the removal rate. As the initial Cd 2+ concentration increases, the removal rate of Cd by CdG01 for Cd 2+ generally shows a downward trend. This may be because at higher concentrations, Cd 2+ increases the toxicity to bacteria, affecting the growth and adsorption ability of bacteria. At lower concentration (25 mg / L), the removal rate of CdG01 is higher, and the removal rate is 96.58% at 192 h, which is the highest. At higher concentration (125 mg / L), the removal rate decreases significantly, indicating that the adsorption ability of CdG01 is limited in a high-concentration Cd 2+ environment. Analysis of the effect of different times on the removal rate found that at all concentrations, the removal rate of Cd by CdG01 2+ gradually increases with time, especially within the first 48 h when the removal rate increases rapidly. After 48 h, the growth rate of the removal rate slows down, indicating that the adsorption ability of CdG01 gradually reaches saturation. At 192 h, the removal rate tends to be stable, indicating that the adsorption ability of CdG01 has basically reached the maximum value at this time point. On the other hand, there are also certain differences in the removal rates at different concentrations. At concentrations of 25 mg / L and 50 mg / L, the removal rates of CdG01 reach 96.58% and 72.61% respectively at 192 h, with relatively high removal rates, indicating that CdG01 has strong adsorption ability at these concentrations. At concentrations of 75 mg / L and 100 mg / L, the removal rates are 62.08% and 51.70% respectively at 192 h, indicating that the adsorption ability has decreased. At a concentration of 125 mg / L, the removal rate is 47.33% at 192 h, indicating that the adsorption ability of CdG01 is significantly reduced at high concentrations.

[0071] Evaluation and analysis of the optimal adsorption ability of the strain found that at low concentration (25 mg / L), CdG01 can reach a relatively high removal rate in a short time (48 h), with high adsorption efficiency and stability, which can provide guidance for the application of Bacillus cereus CdG01 in cadmium pollution control. In actual soil or water body remediation, an appropriate cadmium concentration range can be selected according to the pollution degree to give full play to the adsorption ability of the strain. For example, in an environment with a Cd concentration of about 25 mg / L, the strain CdG01 can achieve the best removal effect and can be considered as a priority application scenario. This result is of great significance for selecting an appropriate cadmium concentration range in practical applications to ensure that the strain can maintain high adsorption ability within a wide concentration range.

[0072] Example 5 Pot experiment on the remediation of Cd-polluted soil by endophytic bacterium CdG01 combined with Vicia villosa

[0073] This example is used to illustrate the remediation of Cd - contaminated soil by the Cd - tolerant endophytic bacterium CdG01 provided by the present invention in combination with Vicia villosa Roth.

[0074] Four treatment groups were set up (Treatment 1: blank control soil; Treatment 2: only Vicia villosa Roth was planted; Treatment 3: only the Bacillus cereus suspension was irrigated into the soil; Treatment 4: Vicia villosa Roth was planted while the Bacillus cereus suspension was irrigated). After thoroughly mixing 8 kg of Cd - contaminated soil, it was filled into polyethylene plastic flowerpots, and each treatment was replicated three times. For the germination and disinfection of Vicia villosa Roth seeds, the seeds were disinfected with 1% sodium hypochlorite solution for 30 min, then rinsed 5 times with sterile water, and then soaked in sterile water at room temperature for 12 h to break the seed dormancy. Then, the Vicia villosa Roth seeds were evenly sown in the flowerpots at a seeding rate of 12 plants per pot in Treatment 2 and Treatment 4, and the planting depth was 1 cm. Subsequently, distilled water was used for irrigation every 3 days to ensure the normal growth of plants. No additional nutrients were added during the growth period of Vicia villosa Roth, and the light, temperature and other conditions of all treatments were kept consistent.

[0075] When the Vicia villosa Roth in the flowerpots grew to a height of 3 cm, the CdG01 bacterial suspension was prepared. The Bacillus cereus CdG01 preserved in glycerol at ultra - low temperature (-80 °C) was activated: the streak plate method was used to culture it on LB solid medium at 30 °C for 24 h. Then, single colonies were picked with a sterile inoculation loop and inoculated into LB liquid medium (300 mL), and cultured at 30 °C with shaking at 200 rpm for 3 days to obtain the strain fermentation broth. Then, the strain fermentation broth was centrifuged to remove the LB liquid medium, and the cell precipitate was retained. The cells were then rinsed clean with sterile water, and finally, the cell precipitate was formulated into a Bacillus cereus suspension of 1×10 8 CFU / mL for standby. After the CdG01 bacterial suspension was prepared, 1 L of the Bacillus cereus suspension with a concentration of 1×10 8 CFU / mL was irrigated into the soil of each treatment in Treatment 3 and Treatment 4, and 1 L of sterile water was irrigated in Treatment 1 and Treatment 2. It was inoculated once a month, and the test period was 3 months, with a total of 3 inoculations. The entire pot experiment was carried out at the experimental base of the Yunnan Academy of Agricultural Sciences. The positions were randomly changed at regular intervals, and during this period, the plant growth conditions such as light and water content of each treatment were kept consistent. Attention should be paid to the prevention and control of plant diseases and insect pests during the growth period of plants, and other field management measures were carried out according to the conventional methods.

[0076] At the end of the test period, Vicia villosa Roth was harvested at the full-bloom stage, and some soil and plant samples were collected. The plant height, root length, number of root nodules, aboveground part, and fresh weight of the roots of each group of plants were recorded, and the heavy metal contents of the aboveground and underground parts were measured. Then, the remaining Vicia villosa Roth plants were cut into pieces and turned over and pressed into the soil in situ for natural decomposition. During this period, the soil moisture management was the same as that during the plant growth period. After 15 days of natural decomposition, the soil was sampled destructively and further analyzed. In addition, according to the above parameters, the blank control and the treatment of only irrigating the Bacillus cereus suspension without planting Vicia villosa Roth were used as controls and sampled at the same time as the plant treatment group.

[0077] 1. Growth of Vicia villosa Roth and its Cd enrichment under different treatments

[0078] This experiment aimed to explore the remediation effect of the cadmium-tolerant endophytic bacterium Bacillus cereus (CdG01) combined with Vicia villosa Roth on cadmium-polluted soil, as well as its effects on the growth and cadmium accumulation characteristics of Vicia villosa Roth, through setting different treatment groups. The experimental results are as Figure 8 shown. From the analysis of the growth status of Vicia villosa Roth, the plant height ( Figure 8 F), root length ( Figure 8 G), aboveground fresh weight ( Figure 8 H), underground fresh weight ( Figure 8 I), and number of root nodules ( Figure 8 J) of treatment 4 (planting Vicia villosa Roth + irrigating the CdG01 suspension) were significantly higher than those of treatment 3 (only planting Vicia villosa Roth, P<0.05), indicating that the CdG01 suspension could improve the overall growth status of the plants. From the perspective of the cadmium enrichment of Vicia villosa Roth, the Cd contents in the aboveground and underground parts ( Figure 8 K) of treatment 4 (planting Vicia villosa Roth + irrigating the CdG01 suspension) were significantly lower than those of treatment 3 (only planting Vicia villosa Roth, P<0.05), indicating that the CdG01 suspension could also reduce the cadmium absorption of Vicia villosa Roth and the accumulation of Cd in the plants.

[0079] In summary, the cadmium-tolerant endophytic bacterium Bacillus cereus (CdG01) combined with Vicia villosa Roth could significantly promote the growth of Vicia villosa Roth, increase its plant height, root length, aboveground and underground fresh weights, and number of root nodules, while reducing the cadmium absorption and accumulation of Vicia villosa Roth. This indicates that the CdG01 suspension has a significant effect on remediating cadmium-polluted soil and can achieve the purpose of remediating cadmium-polluted soil by promoting plant growth and reducing plant cadmium absorption. In addition, the CdG01 suspension could also improve the nitrogen fixation ability of Vicia villosa Roth, further enhancing the plant growth and remediation effect. Therefore, the method for remediating cadmium-polluted soil by the cadmium-tolerant endophytic bacterium Bacillus cereus (CdG01) combined with Vicia villosa Roth provided by the present invention has significant application value and popularization prospects.

[0080] 2. Changes in the content of extractable Cd in soil before and after incorporation

[0081] To explore the remediation efficiency of CdG01 combined with Vicia villosa on Cd - contaminated farmland, the extractable Cd in soil before and after incorporation of Vicia villosa and in soil without Vicia villosa incorporation in different treatments was measured in this study. Among them, DTPA - Cd, also known as available Cd, reflects the bioavailability of Cd in soil. Figure 9 Shows the effect of Vicia villosa incorporation on the content of available cadmium (Cd) in soil. The abscissa represents different treatment groups, including the blank control group (CK), the group only planting Vicia villosa, the group only irrigating with Bacillus cereus suspension, and the group planting Vicia villosa and irrigating with Bacillus cereus suspension simultaneously. The ordinate represents the content of extractable Cd (DTPA - Cd) in soil (unit: mg / kg). Specific analysis found that the content of available Cd in soil of the blank control group (CK) remained basically unchanged before and after incorporation, indicating that the Cd content in soil was stable without any remediation measures. Before incorporation, the content of available Cd in soil of the group only planting Vicia villosa was lower than that of the blank control group, indicating that Vicia villosa had a certain remediation effect on the content of available Cd in soil; after incorporation, the content of available Cd in soil further decreased, indicating that after the residues of Vicia villosa decomposed in soil, it could continuously reduce the bioavailability of Cd in soil. Before incorporation, the content of available Cd in soil of the group only irrigating with Bacillus cereus suspension decreased compared with the blank control group, showing that Bacillus cereus had a certain ability to adsorb and fix Cd, thereby reducing the content of available Cd in soil; after incorporation, the content of available Cd in soil decreased slightly, only by 3.55%, indicating that the remediation ability of Bacillus cereus on soil Cd was limited in a short time, but overall it was still lower than the blank control group before and after incorporation, indicating that Bacillus cereus had a certain persistence in soil and could continuously reduce the activity of Cd in soil. Finally, before incorporation, the content of available Cd in soil of the group planting Vicia villosa and irrigating with Bacillus cereus suspension simultaneously was significantly lower than that of other groups, indicating that the combined remediation effect of Vicia villosa and Bacillus cereus was the best; after incorporation, the content of available Cd in soil further decreased, and the decrease was significantly greater than that of other treatment groups, indicating that the synergistic effect of Vicia villosa and Bacillus cereus could significantly improve the fixation efficiency of Cd in soil and reduce its bioavailability.

[0082] To sum up, Figure 9 It shows that the combined remediation method of Vicia villosa and Bacillus cereus can effectively reduce the bioavailability of Cd in soil, and after the incorporation of Vicia villosa, this reduction effect is more significant. This result verifies the feasibility and effectiveness of this combined remediation method in practical applications, providing strong technical support for the treatment of Cd - contaminated soil.

[0083] Research on the Mitigation of Cd Stress in Successive Cropping Chinese Cabbage by Bacillus cereus Combined with Vicia villosa

[0084] Referring to the seed treatment process in Example 5, transfer the pre-treated Chinese cabbage seeds to a seedling tray filled with soilless substrate and cultivate them at room temperature, while watering irregularly to ensure seed germination. After 2 weeks of seedling cultivation, select Chinese cabbage seedlings with strong growth and uniform growth vigor and transplant them to the soil repaired in Example 5, with a planting density of 3 plants per pot. Before transplantation, apply 15 g of base fertilizer (N:P:K = 18:18:18) to each flower pot, and other field management measures refer to industry standards. The Chinese cabbage is harvested at maturity, and plant samples and soil are collected. Record the plant yield, leaf length, leaf width, number of leaves, and dry weights of the above-ground and underground parts of each group of plants, and measure the heavy metal contents of the above-ground and underground parts.

[0085] Figure 10 The effects of Vicia villosa incorporation on the growth of Chinese cabbage are shown. Blank control group (CK): Each growth index of Chinese cabbage (yield, leaf length, leaf width, number of leaves, above-ground dry weight, underground dry weight) is relatively low, indicating that the growth of Chinese cabbage is restricted to a certain extent without any treatment. The growth indexes of Chinese cabbage in the group only irrigated with Bacillus cereus suspension increased, but the increase range was not large, indicating that the Bacillus cereus suspension has a certain promoting effect on the growth of Chinese cabbage, but the effect is limited. The growth indexes of Chinese cabbage in the group only incorporated with Vicia villosa increased significantly, indicating that the incorporation of Vicia villosa has an obvious promoting effect on the growth of Chinese cabbage. The growth indexes of Chinese cabbage in the group incorporated with Vicia villosa + irrigated with Bacillus cereus suspension are the highest, and are all significantly higher than those of other treatment groups (P<0.05), indicating that the combined treatment of Vicia villosa incorporation and Bacillus cereus suspension has a significant promoting effect on the growth of Chinese cabbage.

[0086] In summary, the combined treatment of Vicia villosa incorporation and Bacillus cereus suspension has a significant promoting effect on the growth of Chinese cabbage, and can significantly increase the yield, leaf length, leaf width, number of leaves, above-ground dry weight, and underground dry weight of Chinese cabbage. Incorporating Vicia villosa alone or irrigating with Bacillus cereus suspension also has a certain promoting effect on the growth of Chinese cabbage, but the effect is not as significant as the combined treatment. The growth of Chinese cabbage in the blank control group is restricted, indicating that the growth environment of Chinese cabbage is not conducive to its normal growth without any treatment. The method for repairing cadmium-polluted soil by the cadmium-tolerant endophytic bacterium Bacillus cereus (CdG01) combined with Vicia villosa provided by the present invention can not only effectively reduce the bioavailability of cadmium in the soil, but also significantly promote the growth of successive crops (such as Chinese cabbage). This method can not only effectively repair cadmium-polluted soil, but also improve the growth and yield of successive crops, and has significant agricultural application value.

[0087] Figure 11It shows the effects of vetch incorporation on cadmium (Cd) accumulation in pakchoi before and after vetch incorporation. Different treatment groups had different effects on the Cd content in the soil. The Cd content in the soil of the blank control group (CK) was relatively high, indicating that without any treatment, the Cd pollution in the soil was relatively serious. The Cd content in the soil of the group only irrigated with Bacillus cereus suspension decreased, indicating that Bacillus cereus suspension had a certain adsorption and fixation effect on Cd in the soil, but the effect was limited. The Cd content in the soil of the group only incorporated with vetch decreased significantly, indicating that the incorporation of vetch could effectively reduce the Cd content in the soil, probably because vetch absorbed some Cd and fixed it in the soil after incorporation. The Cd content in the soil of the group incorporated with vetch + irrigated with Bacillus cereus suspension was the lowest, indicating that the combined treatment of vetch incorporation and Bacillus cereus suspension irrigation could significantly reduce the Cd content in the soil, probably due to their synergistic effect, which enhanced the adsorption and fixation effect on Cd.

[0088] Different treatment groups had different effects on the Cd content in pakchoi. The Cd content in pakchoi of the blank control group (CK) was relatively high, indicating that without any treatment, pakchoi had a strong absorption ability for Cd in the soil, resulting in the accumulation of Cd in the plant. The Cd content in pakchoi of the group only irrigated with Bacillus cereus suspension decreased, but the decrease amplitude was not large, indicating that Bacillus cereus suspension had a certain inhibitory effect on pakchoi's absorption of Cd, but the effect was limited. The Cd content in pakchoi of the group only incorporated with vetch decreased significantly, indicating that the incorporation of vetch could effectively reduce pakchoi's absorption of Cd, probably because vetch absorbed some Cd, reducing the Cd content available for pakchoi to absorb in the soil. The Cd content in pakchoi of the group incorporated with vetch + irrigated with Bacillus cereus suspension was the lowest, indicating that the combined treatment of vetch incorporation and Bacillus cereus suspension irrigation could significantly reduce pakchoi's absorption of Cd, probably due to their synergistic effect, further reducing the Cd content available for pakchoi to absorb in the soil.

[0089] In summary, the combined treatment of incorporating Vicia villosa and applying Bacillus cereus bacterial suspension can significantly reduce the Cd content in soil, decrease the Cd uptake by pakchoi, and thus lower the Cd accumulation in pakchoi. Incorporating Vicia villosa alone or applying Bacillus cereus bacterial suspension alone also has a certain effect on reducing the Cd content in soil and pakchoi, but the effect is not as significant as the combined treatment. The Cd content in pakchoi in the blank control group is relatively high, indicating that without any treatment, pakchoi has a strong ability to absorb Cd from the soil, leading to the accumulation of Cd in the plant, which may pose a threat to food safety. The method for repairing Cd-polluted soil by combining the Cd-tolerant endophytic bacterium Bacillus cereus (CdG01) with Vicia villosa provided by the present invention can not only effectively reduce the bioavailability of Cd in soil, but also significantly reduce the Cd uptake by subsequent crops (such as pakchoi), lower the Cd accumulation in pakchoi, and thus reduce food safety problems caused by excessive Cd. Through the combined treatment of incorporating Vicia villosa and applying Bacillus cereus bacterial suspension, the Cd content in pakchoi is significantly reduced, indicating that this method has a significant inhibitory effect on the Cd uptake of subsequent crops and has important agricultural application value.

[0090] As can be seen from the above examples, the present invention provides a Cd-tolerant Bacillus cereus and its application and method for repairing Cd-polluted soil. The deposit name of the Bacillus cereus is Bacillus cereuss 01, the Latin name is Bacillus cereus, it is deposited in the Guangdong Provincial Microbial Culture Collection Center, the deposit number is GDMCC No: 65570, and the deposit date is December 02, 2024. Through screening and identification, the present invention obtains a highly Cd-tolerant endophytic bacterium. Using this Cd-tolerant endophytic bacterium in combination with Vicia villosa to repair Cd-polluted soil can reduce the Cd uptake by subsequent crops and reduce food safety problems caused by excessive Cd. This method has the advantages of low cost, high repair efficiency, and simple treatment steps.

[0091] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A Cd-resistant Bacillus cereus, characterized in that: The deposited name of the Bacillus cereus is Bacillus cereuss 01, the Latin name is Bacillus cereus, it is deposited in the Guangdong Provincial Microbiological Culture Collection Center, the deposit number is GDMCC No: 65570, and the deposit time is December 2, 2024.

2. Use of the Bacillus cereus described in claim 1 in remediating Cd-contaminated soil.

3. A method for remediating Cd-contaminated soil using the Bacillus cereus according to claim 1, characterized in that: The steps include: (1) sterilizing and soaking the seeds of Vetch, and then sowing them; (2) watering the vetch with the bacterial suspension of Bacillus cereus according to claim 1 after the vetch emerges; (3) Harvest the sweet potato when it reaches the flowering stage and then return the plants to the field.

4. The method according to claim 3, characterized in that The disinfection method in step (1) is as follows: soaking the seeds of Vetch in a 0.8-1.2% sodium hypochlorite solution for 25-35 minutes, followed by rinsing with water for 3-5 times, wherein the soaking time is 10-14 hours, the sowing depth is 0.5-1.2 cm, and watering is performed every 2-4 days after sowing.

5. The method according to claim 4, characterized in that The watering period in step (2) is when the seedlings of Vetch grow to 2.5-3.5 cm, the number of watering is 2-4 times, and the interval between watering is 8-12 days.

6. The method according to claim 5, characterized in that The method for preparing the bacterial suspension in step (2) comprises the following steps: A. inoculating the Bacillus cereus described in claim 1 into LB solid culture medium, and culturing at 28-32° C. for 20-28 h to obtain activated Bacillus cereus; B. Inoculate the activated Bacillus cereus into LB liquid culture medium, and culture at 28-32° C. and 180-220 rpm for 60-84 hours to obtain a fermentation liquid; C. Centrifuge the fermentation broth at 2-6°C for 10-20 minutes to obtain bacterial precipitation; D. Prepare the water for bacterial precipitation to 0.8~1.2×10 8 CFU / mL of bacterial suspension.

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