BSP fungicide as well as preparation method and application thereof

Abstract

The invention provides a BSP microbial inoculum and a preparation method and application thereof, the BSP microbial inoculum is prepared by combining Sargassum koreanum JZ-2 with biochar, and the BSP microbial inoculum is applied to remediation of Cd (II) polluted water and soil and is also applied to improvement of growth stress of Cd (II) on Shanghai green in water culture of Shanghai green. When the concentration of Cd (II) in the Cd (II) polluted water is 5 mg / L, the removal rate of the BSP microbial agent to Cd (II) reaches up to 89.26%. When the concentration of Cd (II) in the Cd (II) polluted soil is 0.25 mg / kg, the fixation rate of the BSP microbial agent on the Cd (II) in the soil is up to 34.78%. By adding the BSP microbial agent, the cadmium stress of the Shanghai Qingqing can be effectively relieved, when the concentration of Cd (II) is 5 mg / L, the fresh weight of the Shanghai Qingqing is increased to 25.15 g from 9.78 g and is increased by 1.57 times, and the Cd (II) enriched in the root system of the Shanghai Qingqing is reduced to 0.8 mg / g from 2.44 mg / g and is reduced by 67.12%. The invention provides a novel method for treating Cd (II) polluted water and soil and relieving the absorption of Cd (II) by Shanghai Qingqing body.

Description

Technical Field

[0001] The present application relates to the technical field of cadmium-contaminated water and soil remediation, and more specifically, to a microbial agent and a preparation method and application thereof, and more specifically, to a BSP agent prepared by combining Korean Bacillus sporesarcinia JZ-2 with biochar and a preparation method and application thereof. Background Art

[0003] Common methods for repairing heavy metal-contaminated water and soil include physical and chemical remediation, phytoremediation, and microbial remediation. Physical and chemical remediation methods have the disadvantages of high cost, high energy consumption, and secondary pollution. Although phytoremediation is low-cost and environmentally friendly, the scarcity of tolerant plants and the unknown mechanism limit its large-scale application. Although the above methods can effectively fix and remove heavy metals in the environment, their disadvantages cannot be ignored.

[0004] Microbial induced carbonate precipitation (MICP) has gradually become an emerging in-situ remediation technology for environmental heavy metal pollution. In the process of microbial induced carbonate precipitation, since the bacterial surface is negatively charged, cations in the solution such as calcium ions and other metal ions can be enriched on the bacterial surface. The metal ions then combine with the carbonate ions produced by the hydrolysis of urea by urease, causing the metal ions to precipitate in the form of carbonates. Microbial induced carbonate precipitation to fix heavy metals has achieved certain results in the laboratory, but large-scale practical applications still need to consider local pH, temperature, bacterial strain type, calcium ion concentration and other influencing factors. Therefore, it is also necessary to explore the mineralization effect in a complex environment and the stability of the metal after fixation. Summary of the invention

[0005] The first object of the present application is to provide a BSP (biochar combined Sporosarcina koreensis JZ-2 pellet) inoculant prepared by combining Korean Sporosarcina JZ-2 with biochar, and its application in fixing Cd(II).

[0006] The second object of the present application is to provide a method for preparing a BSP bacterial agent.

[0007] The third objective of the present application is to provide the application of BSP bacterial agent in the remediation of Cd(II) contaminated water.

[0008] The fourth objective of this application is to provide the application of BSP bacterial agent in the remediation of Cd(II) contaminated soil.

[0009] The fifth objective of the present application is to alleviate the stress of Cd(II) on the growth of Shanghai Grandiflora by adding BSP bacterial agent to Shanghai Grandiflora hydroponics.

[0010] In order to achieve the purpose of the present application, in the first aspect, the present application provides a BSP bacterial agent prepared by combining JZ-2 with biochar and its application in fixing Cd(II).

[0011] In the second aspect, the present application provides a method for preparing a BSP bacterial agent, specifically:

[0012] (1) taking a JZ-2 bacterial solution in the logarithmic growth phase and mixing it with biochar powder to obtain a biochar mixed solution;

[0013] (2) adding the bacterial carbon mixed solution into the sterilized sodium alginate solution to obtain mixed liquid droplets;

[0014] (3) adding the mixed solution dropwise into the CaCl2 solution to form gel particles;

[0015] (4) Place the CaCl2 solution containing the gel particles in a 0-4°C environment for cross-linking for 6 h to 12 h to obtain the BSP bacterial agent, wash it with physiological saline, and store it at 0-4°C for future use.

[0016] Furthermore, in step (1), the addition ratio of JZ-2 bacterial solution to biochar powder is 20 mL:5 g.

[0017] Furthermore, in step (1), the mixing conditions of the JZ-2 bacterial solution and the biochar powder are 30° C. and 150 r / min.

[0018] Furthermore, in step (1), the mixing time of the JZ-2 bacterial solution and the biochar powder is 6 h to 12 h.

[0019] Furthermore, in step (1), the biochar is rice husk or other agricultural waste.

[0020] Furthermore, in step (1), the OD of the JZ-2 bacterial solution in the logarithmic growth phase is 600 It is 0.9~1.2.

[0021] Furthermore, in step (3), the concentration of the CaCl2 solution is 5%.

[0022] Furthermore, the diameter of the BSP inoculant obtained in step (4) is 3-4 mm.

[0023] In a third aspect, the present application provides the application of BSP bacterial agent in the remediation of Cd(II) contaminated water.

[0024] Furthermore, the ratio of the application amount of BSP bacterial agent to Cd(II) contaminated water was 2 g:150 mL.

[0025] Further, the concentration of Cd(II) in the Cd(II) contaminated water is 0-25 mg / L.

[0026] Further, the concentration of Cd(II) in the Cd(II) contaminated water is 5 or 10 or 15 or 20 or 25 mg / L.

[0027] In a fourth aspect, the present application provides the application of BSP bacterial agent in the remediation of Cd(II) contaminated soil.

[0028] Furthermore, the mass ratio of the application amount of BSP bacterial agent to the contaminated soil is 1g:50g.

[0029] Furthermore, the concentration of Cd(II) in the Cd(II) contaminated soil is 0-2 mg / kg.

[0030] Furthermore, the concentration of Cd(II) in the Cd(II) contaminated soil is 0.25 or 0.5 or 1 or 2 mg / kg.

[0031] In a fifth aspect, the present application provides the use of adding BSP bacterial agent in hydroponics of Shanghai Grandiflora to alleviate the growth stress of Cd(II) on Shanghai Grandiflora.

[0032] Furthermore, the ratio of BSP bacterial agent addition to hydroponic solution is 20g:1L.

[0033] Furthermore, the concentration of Cd(II) in the hydroponic solution is 0-10 mg / L.

[0034] Furthermore, the concentration of Cd(II) in the hydroponic solution is 2.5 or 5 or 10 mg / L.

[0035] Through the above technical solution, this application has the following advantages and beneficial effects:

[0036] 1. The Korean Sporosarcina (Sporosarcina koreensis JZ-2, referred to as S.koreensisJZ-2) selected in this application is a patented bacterium previously isolated and purified by the applicant. It has been sequenced and identified by Hefei Fengzhi Biological Company, and the strain sequence has been uploaded to the GenBank database, and its accession number is OM877507. Korean Sporosarcina JZ-2 belongs to the genus Sporosarcina (Sporosarcina Kluyver & Van Niel), is Gram-positive, and strictly aerobic. This application uses MICP as the basic theory, and applies the BSP bacterial agent prepared by combining S.koreensisJZ-2 with biochar to cadmium-contaminated water and soil and to alleviate the enrichment of cadmium in Shanghai green plants. It is a cadmium remediation technology solution with great potential.

[0037] 2. The BSP bacterial agent prepared in this application is in the form of uniform black spheres. As shown in Table 2, the physical and chemical properties of the bacterial agent show that the BSP bacterial agent has good stability and mechanical strength. Figure 4As shown, scanning electron microscopy (SEM) characterization shows that the biochar in the BSP inoculant is tightly bound to the S. koreensis JZ-2 bacteria, proving that the biochar can provide a good living environment for S. koreensis JZ-2.

[0038] 3. Biochar has the advantages of wide raw material sources, low cost, rich pores and functional groups, etc. The growth and development of S.koreensisJZ-2 is easily restricted by the environment, resulting in a decrease in the cadmium remediation effect. Using biochar as a shelter for S.koreensisJZ-2 can better provide a living space for Korean Sporosarcina JZ-2. On the other hand, biochar can provide nutrients for the growth of S.koreensisJZ-2.

[0039] 4. The S. koreensis JZ-22 of the present application has a high tolerance to Cd(II), and the highest tolerance to Cd(II) can reach 30 mg / L.

[0040] 5. The BSP bacterial agent prepared in this application can effectively repair Cd(II) contaminated water, such as Figure 5 As shown, after JZ-2 and biochar were used to make BSP bacterial agent, when the Cd(II) concentration was 5 mg / L, the fixation rate of BSP bacterial agent on Cd(II) was as high as 89.26%.

[0041] 6. The BSP bacterial agent prepared in this application can effectively repair Cd(II) contaminated soil. Figure 6 , 7 As shown in Table 3, after adding BSP, the activities of catalase (S-CAT) and dehydrogenase (sDHA) in the soil increased significantly. The contents of various nutrients in the soil increased significantly. As shown in Table 3, when the Cd(II) concentration was 2 mg / kg, the total nitrogen content of the contaminated soil increased from (3.39±0.52)×10 3 The increase of mg / kg is (7.04±0.70)×10 3 mg / kg, the total phosphorus content increased from 581±50.1mg / kg to 681±35.9mg / kg, the organic matter content in the soil increased from 29.3±4.65g / kg to 40.6±5.18g / kg, and the cation exchange capacity increased from 10.3±0.15mg / kg to 12.7±0.21mg / kg, making the repaired soil more suitable for plant growth. Figure 8As shown in Table 6, when the Cd(II) concentration was 0.25 mg / kg, the fixation rate of BSP bacteria on Cd(II) in the soil was as high as 34.78%; when the Cd(II) concentration was 0.5 mg / kg, the fixation rate of BSP bacteria on Cd(II) in the soil was 27.14%, which was 6.09 times that without adding BSP bacteria.

[0042] 7. Heavy metals have a stress effect on the growth of Shanghai green. By adding the BSP bacterial agent prepared in this application to the nutrient solution containing cadmium, a better fixation effect on Cd(II) can be achieved, effectively alleviating the cadmium stress of Shanghai green. Fig.10 As shown in Figure 2, when the Cd(II) concentration was 5 mg / L, the fresh weight of Shanghai greens increased from 9.78 g to 25.15 g, an increase of 1.57 times. Fig.11 As shown in Table 7, the Cd(II) enrichment in the root system of Shanghai green grass decreased from 2.44 mg / g to 0.8 mg / g, a decrease of 67.12%; when the Cd(II) concentration was 10 mg / L, Fig.11 As shown in Table 7, the Cd(II) enriched in the roots of Shanghai Green decreased from 4.15 mg / g to 3.53 mg / g, a decrease of 14.94%. This shows that some heavy metals were fixed under the action of BSP bacteria and precipitated, which could not be absorbed by plants, thus reducing the toxicity to plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The biomass (OD) of the S. koreensis JZ-2 strain in Example 1 of the present application during its growth under different Cd(II) concentrations in water 600 )change.

[0044] Figure 2 The pH changes during the growth of the S. koreensis JZ-2 strain in Example 1 of the present application under different Cd(II) concentrations in water.

[0045] Figure 3 This is the basic form of the BSP bacterial agent produced in the laboratory in Example 2 of the present application, stored in CaCl2 (A) and after drying (B).

[0046] Figure 4 The morphology and partial magnified image of the BSP bacterial agent under a scanning electron microscope (SEM) in Example 2 of the present application.

[0047] Figure 5 This is the fixation rate of Cd(II) at different concentrations in water by the BSP bacterial agent in Example 2 of the present application.

[0048] Figure 6This is a comparison of the soil catalase (S-CAT) content in the soil before (control group) and after (bacterial agent group) restoration under Cd (II) pollution in Example 3 of the present application. a, b, and c respectively indicate that the differences between the treatments of the bacterial agent group and the control group are significant (p<0.05).

[0049] Figure 7 This is a comparison of the soil dehydrogenase (sDHA) content in the soil before (control group) and after (bacterial agent group) restoration under Cd (II) pollution in Example 3 of the present application. a, b, c, and d respectively indicate that the differences between the treatments of the bacterial agent group and the control group are significant (p<0.05).

[0050] Figure 8 This is a comparison of the heavy metal fixation rate in soil before (control group) and after (bacterial agent group) BSP bacterial agent remediation under Cd (II) pollution in Example 3 of the present application. a, b, c, and d respectively indicate that the differences between the bacterial agent group and the control group are significant (p<0.05).

[0051] Fig. 9 This is the growth morphological changes of Shanghai Grandpa under heavy metal stress in Example 4 of the present application.

[0052] Fig.10 The changes in fresh weight of Shanghai greens without adding bacterial agents (control group: Shanghai greens group) and after adding bacterial agents (experimental group: Shanghai greens + bacterial agent group) in Example 4 of the present application, a and b respectively indicate that the differences between the bacterial agent group and the control group treatment are significant (p<0.05).

[0053] Fig.11 The adsorption of Cd(II) by plant roots (A) and leaves (B) in Example 4 of the present application without adding bacterial agents (control group: Shanghai Qing group) and with adding bacterial agents (experimental group: Shanghai Qing + bacterial agent group), a, b, and c respectively indicate that the differences between the experimental group and the control group are significant (p<0.05). DETAILED DESCRIPTION

[0054] The following examples are used to illustrate the present application, but are not intended to limit the scope of the present application. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0055] Example 1: Tolerance of Korean Sporosarcina JZ-2 (referred to as JZ-2 in this application) to Cd(II)

[0056] Preparation of JZ-2 bacterial suspension

[0057] JZ-2 solid culture medium ingredients: peptone 10g / L, beef extract 3g / L, sodium chloride 5g / L, agar 15g / L, urea 20g / L, pH 7.3±0.2.

[0058] JZ-2 liquid culture medium ingredients: peptone 10g / L, beef extract 3g / L, sodium chloride 5g / L, urea 20g / L, pH 7.3±0.2.

[0059] Experimental method: Under sterile conditions, transfer the JZ-2 (deposit number CGMCC No. 25437) in the above-mentioned JZ-2 solid culture medium to the sterilized JZ-2 liquid culture medium, culture for 24 hours, centrifuge at 4200r / min for 10 minutes, rinse with 0.9% sterile saline for 2 to 3 times, resuspend the JZ-2 bacteria in sterile saline, and prepare a JZ-2 bacterial suspension for use.

[0060] Tolerance of JZ-2 bacterial suspension to different concentrations of Cd(II)

[0061] Take 1 mL of JZ-2 bacterial suspension and add it to 100 mL of sterilized JZ-2 liquid medium with Cd(II) concentration (prepared using Cd(NO3)2·4H2O) of 0, 5, 10, 20, and 30 mg / L, respectively. Culture at 30°C and 130 r / min for 48 h. Samples were taken at 0, 6, 12, 24, 36, and 48 h, and OD was measured. 600 , observe the growth of JZ-2 bacteria.

[0062] The results are as follows Figure 1 As shown, the first 6 hours are the growth adaptation period, with slow growth; 6h-48h are the logarithmic growth period, with rapid growth in number; after 48h, due to the continuous consumption of nutrients, it enters the stable period.

[0063] like Figure 2 As shown, due to the metabolic activity during the growth process, the pH change trend is consistent with the growth trend. When the Cd(II) concentration is 0, 5, 10, and 20 mg / L, the system pH is finally alkaline. When the Cd(II) concentration is 0 mg / L, the system pH is stable at around 9.42.

[0064] Figure 1 and Figure 2 It also reflects the effect of different concentrations of Cd(II) on the growth of JZ-2. As the concentration of Cd(II) increases, the growth of Korean sporosarcina JZ-2 is inhibited. The higher the concentration, the less JZ-2 grows and the later it enters the logarithmic growth phase. When the Cd(II) concentration reaches 30 mg / L, JZ-2 basically stops growing. When the growth of Korean sporosarcina JZ-2 is inhibited, the pH in the culture medium decreases accordingly.

[0065] Example 2: Preparation of BSP bacterial agent and fixation of Cd(II) by BSP bacterial agent

[0066] Preparation of BSP inoculant

[0067] The biochar used in this example is rice husk, which was purchased from the Biochar Research Institute of Zhejiang Academy of Agricultural Sciences.

[0068] Biochar pH determination method: 1.0 g of biochar was taken and diluted with 100 mL of deionized water to prepare a 1% biochar suspension, and the pH value of the solution was measured using a pH meter (Hanna Instruments, Model 3221, USA).

[0069] The total carbon, hydrogen, and nitrogen contents in the biochar were determined by an elemental analyzer (Elementar Vario ELcube, Germany).

[0070] Specific surface area determination: The biochar was ground and passed through a 0.15 mm sieve to calculate its specific surface area using the Brunauer-Emmett-Teller (BET) method.

[0071] The basic physical and chemical properties of biochar are shown in Table 1.

[0072] Table 1 Basic physical and chemical properties of the tested biochar

[0073]

[0074] The rice husk biochar was ground using a mortar and passed through a 100-mesh standard sieve. 20 mL of a suspension of Korean Bacillus sporesarcinia JZ-2 in the logarithmic growth phase (OD 600 =0.9~1.2) is fully mixed with 5g of biochar powder and cultured in a shaker at 30℃ and 150r / min for 12h. Take 4g of sodium alginate and dissolve it in 100mL of distilled water, sterilize it at high temperature and cool it to 40℃. Add the cultured and evenly stirred Korean Sporosarcina JZ-2 and biochar mixture to the sterilized sodium alginate solution, and stir the mixture continuously to mix it evenly. Use a rubber-tipped dropper to add the mixture dropwise to a 5% CaCl2 solution to form gel particles in the CaCl2 solution. Place the CaCl2 solution containing the gel particles in a 0-4℃ refrigerator and cross-link for 6-12h to obtain immobilized bacterial pellet particles (BSP biochar combined Sporosarcina koreensis JZ-2pellet) with a diameter of 3-4mm. Wash it with sterile saline and store it at 4℃ for later use. The morphology of BSP bacterial agent is as follows Figure 3 , Figure 4 shown.

[0075] like Figure 4As shown in the figure, biochar is irregular block with large specific surface area and porous structure. A large number of BSP bacteria can be seen on the surface and pores of BSP bacteria with biochar as carrier. This is mainly because the huge specific surface area and pore structure of biochar provide a good habitat for BSP bacteria. This also shows that biochar is a suitable carrier for making BSP bacteria.

[0076] Determination of diameter and density of BSP bacterial agent: Filter the freshly prepared BSP bacterial agent, wash it with sterile physiological saline, dry it naturally and place it in a measuring cylinder, randomly select 50 particles, weigh them with a 1 / 10,000 electronic balance, measure the mass m of the immobilized bacterial pellets, then add a certain amount of distilled water to completely cover the BSP bacterial agent, measure the volume change, that is, the volume V of 50 BSP bacterial agents, then the density of BSP bacterial agent ρ = m / V. Randomly select 50 BSP bacterial agents of uniform size, measure their diameters with a vernier caliper, and calculate the average diameter.

[0077] pH determination of BSP inoculant: Accurately weigh 0.5 g of BSP and place it in a 20 mL centrifuge tube, then pour in 10 mL of ddH2O, and then place the centrifuge tube in a 30°C constant temperature shaker at a speed of 150 r / min for continuous shaking for 24 hours. Then use a pH meter to measure the pH of the supernatant, that is, the pH of the BSP inoculant. Each BSP inoculant sample was measured in parallel 3 times, and the results were averaged.

[0078] The physicochemical properties of the BSP bacterial agent prepared in this example are shown in Table 2.

[0079] Table 2 Basic physical and chemical properties of BSP microbial agent

[0080] Basic Properties BSP Bacteria Agent Mass (50 pieces) / g 0.87±0.03 <![CDATA[Density / g·cm -1 > 1.17±0.09 Diameter / cm 0.34±0.02 pH 7.1±0.2

[0081] Effect of BSP on the fixation of Cd(II)

[0082] 2 g of BSP inoculant was added to 150 mL of sterilized liquid culture medium with Cd(II) concentrations (prepared using (Cd(NO3)2·4H2O)) of 0, 5, 10, 15, 20, and 25 mg / L, respectively. The culture was incubated at 30°C and 130 r / min for 60 h. Samples were taken every 12 h, and the supernatant was collected and filtered with a 0.22 μm aqueous filter head, and the Cd(II) concentration was determined by flame atomic absorption spectrometry.

[0083] The results of the fixation rate of BSP bacteria on different concentrations of Cd(II) in water are as follows: Figure 5 As shown, BSP has a good fixation ability for Cd(II). When the Cd(II) concentration is 5 mg / L, the fixation rate of BSP for Cd(II) is as high as 89.26%.

[0084] Example 3: Remediation of Cd(II)-contaminated soil by BSP bacterial agent

[0085] The soil samples of Cd(II) contaminated soil in this application were taken from the surface soil (0-20 cm) of Nongcui Garden of Anhui Agricultural University.

[0086] Soil pretreatment: The soil samples were air-dried and ground, then passed through a 100-mesh sieve to remove plant residues and gravel, and then placed in multiple 200 mL beakers for subsequent soil remediation.

[0087] An appropriate amount of cadmium nitrate (Cd(NO3)2·4H2O) was dissolved in 500 mL of deionized water to prepare a cadmium solution, which was then added to 500 g of the pretreated soil sample, mixed and aged thoroughly, and air-dried under natural conditions to obtain the Cd(II) contaminated soil to be repaired. The Cd(II) concentrations were 0, 0.25, 0.5, 1, and 2 mg / kg, respectively.

[0088] The control group consisted of: only adding Cd(II) contaminated soil to be remediated;

[0089] The microbial agent group includes: adding Cd(II) contaminated soil to be repaired and microbial agent, and the addition ratio of microbial agent to Cd(II) contaminated soil to be repaired is 10g:500g.

[0090] After four weeks of cultivation in the laboratory greenhouse, soil samples of the 2 mg / kg control group and the bacterial agent group were taken to determine the composition, nutrients, heavy metal content and enzyme activity in the soil. The results are shown in Table 3.

[0091] The cadmium content in the soil was determined by digestion method. Weigh 0.1000g of ground and sieved air-dried soil sample into a graphite digestion tank, add 4mL of HNO3 and HCl, and leave overnight (10-12h). Heat at 150℃ for 90min, cool for 30min, then add 1mL of HClO4, continue heating at 150℃ for 40min, add 1mL of HNO3 and appropriate amount of distilled water, continue heating until the solution is clear, transfer the solution and dilute to a 25mL volumetric flask, filter with a 0.22μm filter, and determine the cadmium concentration using a Beijing Puxi atomic absorption spectrophotometer (AAS, A3G, China).

[0092] By measuring soil pH, cation exchange capacity (CEC), total nitrogen (TN), total phosphorus (TP), total potassium and organic matter (OM) and other indicators, the changes in soil physical and chemical properties and fertility before and after the restoration of this application were shown.

[0093] Soil pH was determined using deionized water as the extractant with a water-to-soil ratio of 2.5 to 1, and then a pH meter (HJ 962-2018) was used to determine the pH; the cation exchange capacity was determined using the ammonium acetate exchange method (LY / T1243-1999); the soil total nitrogen was determined using the Kjeldahl method (HJ 717-2014); the soil total phosphorus was determined using the alkali fusion-molybdenum antimony counterspectrophotometry method (HJ632-2011); the soil total potassium was determined using the soil total potassium determination method (NY / T 87-1988); and the soil organic matter was determined using the potassium dichromate oxidation-external heating method (LY / T 1237-1999).

[0094] By measuring indicators such as soil catalase (S-CAT) activity and dehydrogenase (sDHA) activity, the ability of soil enzymes to eliminate the toxic effects of hydrogen peroxide (H2O2) in the soil and the degradation ability of soil microorganisms before and after restoration using the bacterial agent of the present application are shown.

[0095] The kit is used to measure the activity of soil catalase (S-CAT) in the soil. H2O2 has a characteristic absorption peak at 240nm. By measuring the change in absorbance of the solution at this wavelength after reacting with the soil, the level of S-CAT activity can be reflected.

[0096] The activity of soil dehydrogenase (sDHA) in soil was determined using a kit. After the hydrogen acceptor 2,3,5-triphenyl tetrazolium chloride (2,3,5-Triphenyl Tetrazolium Chloride.TTC) accepts hydrogen during cellular respiration, it is reduced to triphenyl formazore (TriphenyI Formazore, TF), which has a maximum absorption peak at a wavelength of 485nm. The absorbance value at 485nm is measured by spectrophotometry to obtain the soil dehydrogenase activity.

[0097] Calculation of Cd(II) removal efficiency (Cd(II) fixation rate):

[0098]

[0099] Where: R is the removal rate of Cd(II) (%), C0 is the initial concentration of Cd(II) (mg / L), C t is the residual concentration of Cd(II) (mg / L).

[0100] Origin 2021 software and Excel 2019 were used for data processing and plotting, and XRD was analyzed using MDI Jade 6 software.

[0101] Table 3 Changes in soil physical and chemical properties and fertility before and after restoration of this application (Cd(II) concentration 2 mg / kg)

[0102]

[0103] Soil catalase (S-CAT) is mainly derived from the secretions of soil microorganisms and plant roots. It is an important enzyme in soil biological metabolism. Its main function is to eliminate the toxic effects of H2O2 in the soil and plays an important role in the active oxygen scavenging system. Figure 6 As shown in Table 4, in the control group, under heavy metal pollution, the activity of catalase (S-CAT) in the soil decreased with the increase of heavy metal concentration; while in the bacterial agent group, due to the addition of BSP bacterial agent, the enzyme activity increased compared with the control group.

[0104] Table 4 Summary of S-CAT activity data of control group and BSP bacterial agent group with changes in Cd(II) concentration

[0105]

[0106] Soil dehydrogenase (sDHA) activity can reflect the amount of active microorganisms in the soil system and their degradation activity of organic matter, and can be used as an important indicator to judge the degradation performance of soil microorganisms. Figure 7 As shown in Table 5, it can be clearly seen that compared with the control group, the soil dehydrogenase activity of the BSP bacterial agent group was significantly improved.

[0107] Table 5 Summary of sDHA enzyme activity data of the control group and BSP bacterial agent group with changes in Cd(II) concentration

[0108]

[0109] like Figure 8 As shown in Table 6, when the Cd(II) concentration was 0.25 mg / kg, the fixation rate of Cd(II) in the soil by the BSP inoculum group was as high as 34.78%; when the Cd(II) concentration was 0.5 mg / kg, the fixation rate of Cd(II) in the BSP inoculum group was 27.14%, while that of the control group was only 3.83%. The fixation rate of the BSP inoculum group was 6.09 times that of the control group. This shows that the BSP inoculum has a good fixation effect on Cd(II).

[0110] Table 6 Summary of Cd(II) fixation rate (%) in control group and BSP bacterial agent group as Cd(II) concentration changes

[0111]

[0112] Example 4: Experiment on reducing cadmium absorption by hydroponic broccoli using BSP bacterial agent

[0113] Experimental materials: One-year-old Shanghai green, purchased from Shouguang Xinxinran Horticulture Co., Ltd.

[0114] The nutrient solution used for the tested bok choy was semi-Hoagland nutrient solution.

[0115] The preparation method of BSP bacterial agent is shown in Example 2.

[0116] Control group: Take an equal amount of Shanghai green plants that have been cultured in the laboratory greenhouse soil for two weeks and place them in a nutrient solution with Cd(II) concentrations of 0, 2.5, 5, 10, and 20 mg / L for hydroponics. After four weeks, take out the whole Shanghai green plants cultured in hydroponics and measure their fresh weight and cadmium content in the plants.

[0117] Experimental group: An equal amount of Shanghai green plants cultured in the laboratory greenhouse for two weeks were placed in 1L nutrient solution with Cd(II) concentrations of 0, 2.5, 5, 10, and 20 mg / L for hydroponics. 20 g of the BSP inoculum prepared in Example 2 was added to the nutrient solution respectively. After four weeks of cultivation, the whole Shanghai green plants cultured in water were taken out to determine their fresh weight and cadmium content in the plants.

[0118] Determination of Cd(II) content in plants: The digestion method was used to determine the Cd(II) content in plants. Weigh 0.02 g of fresh and clean Shanghai green leaves / roots, cut them into filaments with a length of about 5 mm and a width of about 1 mm, put them into a graphite digestion tube, add 1 mL of HClO4 and 9 mL of concentrated HNO3, and leave them overnight (10 to 12 h). In a graphite digestion instrument, digest at 100°C for 10 minutes, 150°C for 10 minutes, and continue digesting at 180°C until the solution is clear. Transfer the solution and adjust the volume to 50 mL, filter it with a 0.22 μm filter head, take the filtrate, and determine the Cd(II) content using a ZEEnit700P atomic absorption spectrophotometer (Jena, Germany).

[0119] The toxicity of Cd(II) can have a significant impact on plant growth. Fig. 9 As shown in the figure, after adding Cd(II), the leaves of the Shanghai green plants turned yellow, the plants became smaller, and the rhizomes became soft. Compared with the control group, the addition of low concentrations of Cd(II) promoted the growth of the plants. Fig. 9 As shown in the figure, when 2.5 mg / L Cd(II) was added, the plants became larger and the chlorophyll content in the leaves increased. However, when the Cd(II) concentration exceeded 2.5 mg / L, the plants gradually turned yellow and became soft, and the mortality rate also gradually increased.

[0120] Depend on Fig.10 As shown in the figure, the change in the fresh weight of Shanghai green after adding BSP bacteria can show that BSP bacteria has a strong promoting effect on the growth of Shanghai green, and this effect reaches the maximum at 5 mg / L. Fig.10It can be seen that after adding BSP bacteria (experimental group), the fresh weight of Shanghai green more than doubled compared with that without adding BSP bacteria (control group), up to 1.57 times, indicating that the addition of BSP bacteria effectively fixed Cd(II) in the nutrient solution and reduced the enrichment of Cd(II) in the plant body, thereby alleviating the Cd(II) stress of Shanghai green.

[0121] like Fig.11 As shown in Table 7, after adding BSP bacterial agent to the culture medium with a concentration below 10 mg / L, the heavy metal content in the plant body decreased significantly. When the Cd(II) concentration was 5 mg / L, the Cd(II) enriched in the root system of Shanghai Qingqing decreased from 2.44 mg / g (control group) to 0.8 mg / g (experimental group), a decrease of 67.21%; when the Cd(II) concentration was 10 mg / L, the Cd(II) enriched in the root system of Shanghai Qingqing decreased from 4.15 mg / g (control group) to 3.53 mg / g (experimental group), a decrease of 14.94%, indicating that some heavy metals were fixed under the action of BSP bacterial agent, resulting in precipitation, which could not be absorbed by plants, thereby reducing the toxicity of plants.

[0122] Table 7 Data summary of Cd(II) concentrations in roots and leaves of Shanghai Qing group and Shanghai Qing + bacterial agent group

[0123]

[0124] Although the present application has been described in detail above with general descriptions and specific implementation schemes, it is obvious to those skilled in the art that some modifications or improvements may be made to the present application. Therefore, these modifications or improvements made without departing from the spirit of the present application all fall within the scope of protection claimed in the present application.

Claims

1. A BSP bacterial agent, characterized in that: It is prepared by combining Korean Bacillus sporesarcinia JZ-2 with biochar.

2. The BSP bacterial agent according to claim 1, characterized in that: The application of the BSP bacterial agent in fixing Cd(II).

3. The BSP bacterial agent according to claim 1, characterized in that: The preparation method of the BSP bacterial agent is: The JZ-2 bacterial suspension in the logarithmic growth phase is fully mixed with the biochar powder to obtain a biochar mixed solution; Adding the bacterial carbon mixed solution into the sterilized sodium alginate solution to obtain mixed liquid drops; adding the mixed solution dropwise into a CaCl2 solution to form gel particles; The CaCl2 solution containing the gel particles is placed in a 0-4°C environment for cross-linking for 6-12 hours to obtain the BSP bacterial agent.

4. The BSP bacterial agent according to claim 3, characterized in that: The OD of the JZ-2 bacterial suspension in the logarithmic growth phase is 600 It is 0.9~1.

2.

5. The BSP bacterial agent according to claim 1 or 2, characterized in that: The application of the BSP bacterial agent in the remediation of Cd(II) contaminated water.

6. The BSP bacterial agent according to claim 5, characterized in that: The concentration of Cd(II) in the Cd(II)-contaminated water is 0-25 mg / L.

7. The BSP bacterial agent according to claim 1 or 2, characterized in that: The application of the BSP bacterial agent in the remediation of Cd(II) contaminated soil.

8. The BSP bacterial agent according to claim 7, characterized in that: The concentration of Cd(II) in the Cd(II) contaminated soil is 0-2 mg / kg.

9. The BSP bacterial agent according to claim 1 or 2, characterized in that: The BSP bacterial agent is added to the Shanghai Fern hydroponic solution to alleviate the stress of Cd(II) on the growth of Shanghai Fern.

10. The BSP bacterial agent according to claim 9, characterized in that: The concentration of Cd(II) in the hydroponic solution is 0-10 mg / L.

Images