Bacteria capable of accumulating heavy metals, and methods of making and using the same

Abstract

The application discloses a kind of bacteria that can enrich heavy metal and its preparation method and application, and the bacteria classification name is Aestuariibaculum sp.JKB11, is preserved in China Microorganism Strain Preservation Management Committee general microorganism center, and the preservation number is CGMCC No.27739.The bacteria can effectively enrich Cd 2+ , Ni 2+ , Cu 2+ And Zn 2+ , has potential repair effect in cadmium, nickel, copper, zinc pollution environment.Meanwhile, the bacteria can be prepared into microbial inoculant by immobilization to realize the producibility in practical application, low cost, simple operation has recyclability, environmental protection is efficient, and secondary pollution is not easy to produce, provides greater possibility and operability for realizing the removal of heavy metal ions in polluted water body.

Description

Technical Field

[0001] This invention relates to the field of microbial development and utilization technology, and in particular to a bacterium capable of accumulating heavy metals, its preparation method, and its application. Background Technology

[0002] Due to rapid agricultural and industrial development, indiscriminate discharge of industrial wastewater, improper treatment of industrial waste, and excessive use of fertilizers and pesticides, large amounts of heavy metal pollutants have entered the environment. Heavy metals are carcinogenic, teratogenic, and mutagenic, causing extremely adverse effects on the ecological environment. They can also accumulate through the food chain and food web, entering the human body and harming human health. Currently, increasingly serious heavy metal pollution has become a major environmental safety problem seriously affecting the ecological environment and human health, urgently requiring effective methods for its treatment.

[0003] Traditional methods for treating heavy metal pollution are mostly physical and chemical methods, such as chemical precipitation, ion exchange, reverse osmosis, and activated carbon adsorption. These methods can effectively remove heavy metals, but they also have problems to varying degrees, such as complex operation, high cost, high energy consumption, and easy generation of secondary pollution.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a bacterium capable of enriching heavy metals, its preparation method and application, aiming to solve the problems of complex operation, high cost and easy secondary pollution that exist to varying degrees in the existing physical and chemical methods for treating heavy metal pollution.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a bacterium capable of accumulating heavy metals, wherein the bacterium is classified and named as follows: Aestuariibaculum sp. JKB11, the bacteria described is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 27739.

[0007] Optionally, the heavy metal includes at least one of cadmium, nickel, copper, and zinc.

[0008] A second aspect of the present invention provides an application of the bacteria capable of accumulating heavy metals as described above, wherein the bacteria capable of accumulating heavy metals are used to accumulate heavy metals.

[0009] Optionally, the bacteria can be immobilized to form a microbial agent for enrichment of at least one of cadmium, nickel, copper, and zinc.

[0010] The third invention provides a microbial agent comprising a carrier and bacteria loaded on the carrier, wherein the bacteria are the heavy metal-accumulating bacteria described above.

[0011] Optionally, the carrier is biochar; or, the carrier comprises sodium alginate and polyvinyl alcohol.

[0012] A fourth aspect of the present invention provides a method for preparing a microbial inoculant, comprising the steps of: loading bacteria onto a carrier to obtain the microbial inoculant; wherein the bacteria are the heavy metal-accumulating bacteria of the present invention as described above.

[0013] Optionally, the preparation method of the microbial agent specifically includes the following steps; The bacteria capable of accumulating heavy metals were added to a culture medium containing biochar, cultured, and then centrifuged to obtain the microbial inoculum.

[0014] Optionally, the preparation method of the microbial agent specifically includes the following steps: A mixed solution containing sodium alginate and polyvinyl alcohol is provided; The bacteria capable of accumulating heavy metals are added to the mixed solution to form a microbial inoculant solution; The microbial agent solution is added dropwise to a mixed solution of boric acid and CaCl2 to obtain the microbial agent.

[0015] Beneficial effects: This invention screened bacteria with heavy metal tolerance from the tidal flats of the Futian Mangrove Nature Reserve in Shenzhen. Based on 16S rDNA sequence alignment, whole-genome sequencing, and calculated ANI and DNA-DNA hybridization values, it was found that… Aestariibaculum A potential new fungus within the genus was identified as Aestuariibaculum sp. JKB11. Aestuariibaculum sp. JKB11 can effectively enrich Cd in solution. 2+ Ni 2+ Cu 2+ and Zn 2+ In Cd containing 10 mg / L, 2+ Ni 2+ Cu 2+ Zn 2+ In solutions containing heavy metal ions Cd 2+ Ni 2+ Cu 2+ Zn 2+ The removal rates reached 91.03%, 75.38%, 82.24%, and 90.00%, respectively, at concentrations of 50 mg / L Cd. 2+ Ni 2+ Cu 2+ Zn2+ In solutions containing heavy metal ions Cd 2+ Ni 2+ Cu 2+ Zn 2+ The removal rates reached 22.02%, 14.09%, 32.30%, and 18.90%, respectively, and the adsorption capacities reached 8.43 mg / g, 5.86 mg / g, 11.50 mg / g, and 7.67 mg / g, respectively. When... Aestuariibaculum sp. After JKB11 was immobilized and prepared into a microbial inoculum, it was subjected to treatment with 50 mg / L Cd. 2+ Ni 2+ Cu 2+ Zn 2+ In solutions containing heavy metal ions Cd 2+ Ni 2+ Cu 2+ Zn 2+ The removal rates reached 49.80%, 56.60%, 73.47%, and 58.01%, respectively, and the adsorption capacities reached 30.54 mg / g, 23.94 mg / g, 27.01 mg / g, and 23.52 mg / g, respectively. This indicates... Aestariibaculum sp. JKB11 has potential remediation effects in environments contaminated with cadmium, nickel, copper, and zinc. The bacteria provided by this invention are characterized by low cost, low energy consumption, simple operation, recyclability, environmental friendliness, high efficiency, and low likelihood of secondary pollution when treating heavy metal ions, thus facilitating the removal of Cd from polluted water. 2+ Ni 2+ Cu 2+ and Zn 2+ It offers greater possibilities and operability. Meanwhile, Aestuariibaculum sp. JKB11 can produce a large amount of microbial biomass through fermentation in a short time. This invention utilizes immobilization... Aestuariibaculum sp. The microbial agent prepared using JKB11 has achieved its producibility for practical applications. This invention aims to study the utilization of microorganisms in environments contaminated with cadmium, nickel, copper, and zinc. Aestuariibaculum sp. JKB11 lays the foundation for remediation, enriches the microbial library for using microorganisms to remediate heavy metal pollution, and provides technical support for the ultimate realization of using microorganisms to remediate heavy metal pollution in water and soil. Attached Figure Description

[0016] Figure 1 The bacteria capable of accumulating heavy metals obtained in Example 1 of this invention Aestuariibaculum sp. Photograph of JKB11 on a plate.

[0017] Figure 2 In Embodiment 1 of the present invention Aestuariibaculum sp. JKB11 Neighbor-Joining phylogenetic tree constructed based on 16S rDNA gene sequence.

[0018] Figure 3 The images show actual bacterial solutions with different biochar concentrations in Example 3 of this invention.

[0019] Figure 4 This is a physical image of the PVA-SA microbial agent in Example 4 of the present invention.

[0020] Figure 5 In (a), different biochar concentrations of biochar-microbial inoculants were used in Example 5 of the present invention at a concentration of 50 mg / L Cd. 2+ Cd in solution 2+ The results of removal rate and adsorption amount are shown in Figure (b). (b) shows the biochar-microbial agent with different biochar concentrations in Example 5 of the present invention at 50 mg / L Ni. 2+ Ni in solution 2+ The results of removal rate and adsorption capacity are shown in Figure (c), which shows the biochar-microbial agent with different biochar concentrations in Example 5 of this invention at 50 mg / L Cu. 2+ Cu in solution 2+ The graph shows the removal rate and adsorption capacity results. (d) shows the biochar-microbial agent with different biochar concentrations in Example 5 of this invention at 50 mg / L Zn. 2+ Zn in solution 2+ The results of removal rate and adsorption amount are shown in the figure.

[0021] Figure 6 In Embodiment 5 of the present invention Aestariibaculum sp. JKB11 and the biochar-microbial inoculant with the optimal biochar content were respectively used at 50 mg / L Cd. 2+ Ni 2+ Cu 2+ Zn 2+ The results of the removal rate and adsorption amount of heavy metal ions in the solution are shown in the figure.

[0022] Figure 7 The different raw material ratios of PVA-SA-microbial agents in Example 5 of this invention are used in a 50 mg / L Cd solution. 2+ Cd in solution 2+ The results of removal rate and adsorption amount are shown in Figure (b). (b) shows the PVA-SA-microbial agent with different raw material ratios in Example 5 of the present invention at 50 mg / L Ni. 2+ Ni in solution 2+ The results of removal rate and adsorption capacity are shown in Figure (c), which shows the PVA-SA-microbial agent with different raw material ratios in Example 5 of the present invention at 50 mg / L Cu. 2+Cu in solution 2+ The graph shows the removal rate and adsorption capacity results. (d) shows the PVA-SA-microbial agent with different raw material ratios in Example 5 of this invention at 50 mg / L Zn. 2+ Zn in solution 2+ The results of removal rate and adsorption amount are shown in the figure.

[0023] Figure 8 In Embodiment 5 of the present invention Aestariibaculum sp. JKB11 and the optimal raw material ratio of PVA-SA-microbial agent were respectively used at 50 mg / L Cd. 2+ Ni 2+ Cu 2+ Zn 2+ The results of the removal rate and adsorption amount of heavy metal ions in the solution are shown in the figure. Detailed Implementation

[0024] This invention provides bacteria capable of accumulating heavy metals, a method for preparing the bacteria, and their applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0026] Mangroves are ecosystems transitioning from land to sea, rich in biological resources. In recent years, with rapid economic and industrial development, mangroves, as natural barriers along the coast, have accumulated large amounts of pollutants from human activities, including heavy metals. Heavy metals are characterized by long residual times, reluctance to degrade, and easy bioaccumulation through the food chain, leading to long-term heavy metal stress on microorganisms in mangrove areas. Since microorganisms themselves possess certain environmental specificities and follow the natural law of survival of the fittest in biological evolution, some microorganisms can develop a certain degree of heavy metal tolerance in heavily polluted environments, and even utilize heavy metals to complete certain physiological activities. These microorganisms have become the dominant heavy metal-tolerant populations in mangrove areas. Therefore, mangroves are an excellent environment for cultivating microorganisms that can tolerate heavy metal growth. Based on this, this invention uses soil from the tidal flats of the Shenzhen Futian Mangrove Nature Reserve as material. Through selective domestication and isolation of native microorganisms with heavy metal tolerance, and immobilization treatment, this invention deeply explores their ability to accumulate heavy metals, enriching the microbial reservoir for heavy metal remediation and providing technical support for future practical applications of microbial remediation of heavy metal pollution. Specifically, this embodiment of the invention provides a bacterium capable of accumulating heavy metals, wherein the bacterium is classified and named as follows: Aestuariibaculum sp. JKB11, the bacteria described therein, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on July 5, 2023, with accession number CGMCC No. 27739.

[0027] This invention screened bacteria with heavy metal tolerance from the tidal flats of the Futian Mangrove Nature Reserve in Shenzhen. Based on 16S rDNA sequence alignment, whole-genome sequencing, and calculated ANI and DNA-DNA hybridization values, it was found that… Aestariibaculum A potential new fungus within the genus was identified as Aestuariibaculum sp. JKB11. Aestuariibaculum sp. The optimal growth temperature for JKB11 is 28°C, the optimal salinity is 30 ppt, and the optimal pH is 7.0. Aestuariibaculum sp. JKB11 can effectively enrich Cd in solution. 2+ Ni 2+ Cu 2+ and Zn 2+ In Cd containing 10 mg / L, 2+ Ni 2+ Cu 2+ Zn 2+ In solutions containing heavy metal ions Cd 2+ Ni 2+ Cu 2+ Zn 2+The removal rates reached 91.03%, 75.38%, 82.24%, and 90.00%, respectively, in Cd concentrations of 50 mg / L. 2+ Ni 2+ Cu 2+ Zn 2+ In solutions containing heavy metal ions Cd 2+ Ni 2+ Cu 2+ Zn 2+ The removal rates reached 22.02%, 14.09%, 32.30%, and 18.90%, respectively, and the adsorption capacities reached 8.43 mg / g, 5.86 mg / g, 11.50 mg / g, and 7.67 mg / g, respectively. When... Aestariibaculum sp. After JKB11 was immobilized and prepared into a microbial inoculum, it was subjected to treatment with 50 mg / L Cd. 2+ Ni 2+ Cu 2+ Zn 2+ In solutions containing heavy metal ions Cd 2+ Ni 2+ Cu 2+ and Zn 2+ The removal rates reached 49.80%, 56.60%, 73.47%, and 58.01%, respectively, and the adsorption capacities reached 30.54 mg / g, 23.94 mg / g, 27.01 mg / g, and 23.52 mg / g, respectively. This indicates... Aestuariibaculum sp. JKB11 has potential remediation effects in environments contaminated with cadmium, nickel, copper, and zinc. The bacteria provided by this invention are characterized by low cost, low energy consumption, simple operation, recyclability, environmental friendliness, high efficiency, and low likelihood of secondary pollution when treating heavy metal ions, thus facilitating the removal of Cd from polluted water. 2+ Ni 2+ Cu 2+ and Zn 2+ It offers greater possibilities and operability. Meanwhile, Aestuariibaculum sp. JKB11 can produce a large amount of microbial biomass through short-term fermentation. This invention utilizes immobilized bacteria... Aestuariibaculum sp. The microbial agent prepared by JKB11 has achieved producibility for practical applications. This invention aims to study the utilization of microorganisms in environments contaminated with cadmium, nickel, copper, and zinc. Aestuariibaculum sp. JKB11 lays the foundation for remediation, enriches the microbial library for using microorganisms to remediate heavy metal pollution, and provides technical support for the ultimate realization of using microorganisms to remediate heavy metal pollution in water and soil.

[0028] In some embodiments, the heavy metal includes at least one of cadmium, nickel, copper, and zinc.

[0029] This invention also provides an application of the bacteria capable of accumulating heavy metals as described above, wherein the bacteria capable of accumulating heavy metals are used to accumulate heavy metals.

[0030] The bacteria can be produced in environments containing heavy metal ions Cd. 2+ Ni 2+ Cu 2+ and Zn 2+ Grown on solid culture medium, for Cd 2+ Ni 2+ Cu 2+ and Zn 2+ The tolerance levels were 150 mg / L, 400 mg / L, 0 mg / L, and 0 mg / L, respectively. The bacteria exhibited tolerance to the heavy metal ion Cd. 2+ Ni 2+ Cu 2+ and Zn 2+ The bacteria exhibit high removal capacity at 50 mg / L Cd. 2+ When cultured in solution, Cd 2 + The removal rate reached 22.02%, and the enrichment amount (or adsorption amount, removal amount) was 8.43 mg / g; the bacteria were in 50 mg / L Ni 2+ When cultured in solution, for Ni 2+ The removal rate reached 14.09%, and the enrichment amount was 5.86 mg / g; the bacteria were enriched in 50 mg / L Cu. 2+ When culturing in solution, for Cu 2+ The removal rate reached 32.30%, and the enrichment amount was 11.50 mg / g. The bacteria were in a solution of 50 mg / L Zn. 2+ When cultured in solution, for Zn 2+ The removal rate reached 18.90%, and the enrichment amount was 7.67 mg / g. The bacteria were subjected to treatment at 10 mg / L Cd. 2+ Ni 2+ Cu 2+ Zn 2+ When cultured in solution, Cd 2+ Ni 2+ Cu 2+ Zn 2+ The removal rates reached 91.03%, 75.38%, 82.24%, and 90.00%, respectively.

[0031] Bioremediation is an innovative technology for removing and recovering heavy metal ions from polluted areas. It involves utilizing the activity of algae, bacteria, fungi, or the plants themselves, using living organisms to reduce or restore heavy metal pollutants to less harmful forms. Bioremediation is characterized by its simplicity, low cost, and environmental friendliness, making it a current research hotspot in heavy metal remediation. It has been used to remove heavy metals from contaminated wastewater and soil, and this superior method can replace physical and chemical methods. While microbial remediation offers advantages such as low cost, greenness, and high efficiency, it also has certain limitations. The industrial application of bioremediation technology is restricted by two main factors: a lack of long-term operational stability and difficulty in recovering and reusing cells. Immobilization of microorganisms, by chemically or physically restricting cell mobility while providing cell protection, effectively overcomes these drawbacks. Cell immobilization technology is increasingly important in wastewater treatment because, compared to using free cells, it offers advantages such as high biomass, reusable cells, high mechanical strength, strong resistance to toxic chemicals, improved genetic stability, and elimination of cell flushing problems. Based on this, in some embodiments, the bacteria are immobilized to form microbial agents for the enrichment of cadmium, nickel, copper, and zinc. The immobilized microbial agents prepared in actual heavy metal-contaminated wastewater exhibit better precious metal enrichment effects compared to single strains (i.e., unimmobilized strains).

[0032] Furthermore, embodiments of the present invention also provide a microbial agent, comprising a carrier and bacteria loaded on the carrier, wherein the bacteria are the heavy metal-accumulating bacteria described above in embodiments of the present invention.

[0033] In some embodiments, the carrier is biochar (e.g., corn stalk biochar). Biochar itself has a certain physical adsorption capacity for heavy metals, and when combined with the heavy metal-accumulating bacteria described above in the embodiments of the present invention at a certain ratio, the resulting microbial agent has an even higher removal capacity for heavy metals. (The last sentence appears to be incomplete and possibly refers to a separate experiment involving 50 mg / L Cd.) 2+ Ni 2+ Cu 2+ Zn 2+ In solutions containing biochar, microbial agents have an effect on the heavy metal ion Cd. 2+ Ni 2+ Cu 2+ Zn 2+ The removal rates reached 36.07%, 22.58%, 37.62%, and 25.62%, respectively, for heavy metal ions Cd. 2+ Ni 2+ Cu 2+ and Zn 2+The adsorption capacities can reach 10.49 mg / g, 6.65 mg / g, 10.96 mg / g, and 7.90 mg / g, respectively.

[0034] In some embodiments, the carrier includes sodium alginate and polyvinyl alcohol. In this embodiment, after immobilizing the bacteria capable of accumulating heavy metals into gel spheres using sodium alginate and polyvinyl alcohol as carriers, thus forming a microbial agent, the heavy metal ion Cd is effectively contained. 2+ Ni 2+ Cu 2+ and Zn 2+ It exhibits higher removal capacity than unimmobilized strains, in Cd concentrations of 50 mg / L. 2+ Ni 2+ Cu 2+ Zn 2+ In solutions containing sodium alginate and polyvinyl alcohol, microbial agents have an effect on the heavy metal ion Cd. 2+ Ni 2+ Cu 2+ Zn 2+ The removal rates reached 49.80%, 56.60%, 73.47%, and 58.01%, respectively, for heavy metal ions Cd. 2+ Ni 2 + Cu 2+ and Zn 2+ The adsorption capacities can reach 30.54 mg / g, 23.94 mg / g, 27.01 mg / g, and 23.52 mg / g, respectively.

[0035] In this embodiment, the immobilization of bacteria with sodium alginate and polyvinyl alcohol provides protection for the bacterial strain, reduces the toxic effects of heavy metals on the strain, and improves the bacteria's ability to remove heavy metals. The optimal mass ratio of sodium alginate to polyvinyl alcohol for immobilized bacteria is 14 parts polyvinyl alcohol and 1 part sodium alginate. Aestuariibaculum sp. JKB11 (0.288 parts) and high-purity water (0.13 parts) showed that the removal capacity of microbial agents for heavy metals first increased and then decreased with the increase of bacterial count. This may be because within a certain range of bacterial count, the removal capacity of microbial agents for heavy metals increases with the increase of bacterial count, but after exceeding a certain amount, the microsphere structure of the microbial agents becomes unstable and difficult to form, and the removal capacity also decreases.

[0036] In this embodiment, Aestuariibaculum sp. JKB11 can produce a large amount of microbial biomass through short-term fermentation. The microbial agent prepared from sodium alginate and polyvinyl alcohol is feasible for practical applications, offering low cost, simple operation, recyclability, and environmental friendliness. It is highly efficient and environmentally friendly, facilitating the removal of Cd from polluted water. 2+Ni 2+ Cu 2+ and Zn 2+ This provides greater possibilities and operability. Specifically, the microbial agent is in the form of small spheres, exhibiting better mechanical strength than unimmobilized strains and is easier to recover. The microbial agent prepared by immobilization with sodium alginate and polyvinyl alcohol is simple to operate in actual heavy metal-contaminated wastewater treatment, is recyclable, and environmentally friendly, thus facilitating the removal of Cd from polluted water. 2+ Ni 2+ Cu 2+ and Zn 2+ It offers greater possibilities and operability.

[0037] This invention also provides a method for preparing a microbial agent, comprising the steps of: loading bacteria onto a carrier to obtain the microbial agent; wherein the bacteria are the heavy metal-accumulating bacteria described above in this invention.

[0038] In some embodiments, the preparation method of the microbial agent specifically includes the following steps; The bacteria capable of accumulating heavy metals were added to a culture medium containing biochar, cultured, and then centrifuged to obtain the microbial inoculum.

[0039] In some embodiments, the preparation method of the microbial inoculant specifically includes the following steps: A mixed solution containing sodium alginate and polyvinyl alcohol is provided; The bacteria capable of accumulating heavy metals are added to the mixed solution to form a microbial agent solution; The microbial agent solution is added dropwise to a mixed solution of boric acid and CaCl2 to obtain the microbial agent.

[0040] The following detailed description uses specific examples.

[0041] Unless otherwise specified, all raw materials used in the following examples are commercially available products.

[0042] The following examples illustrate the formulation of R2A solid culture medium: Yeast Extract: 0.5 g / L; Peptone: 0.5 g / L; Casein Hydrolysate: 0.5 g / L; Dextrose: 0.5 g / L; Soluble Starch: 0.5 g / L; Dipotassium Phosphate: 0.3 g / L; Magnesium Sulfate: 0.024 g / L; Sodium Pyruvate: 0.3 g / L; Agar: 15.0 g / L; pH: 7.2 ± 0.2.

[0043] Example 1 Aestuariibaculum sp. Isolation and Identification of JKB11 (1) Soil sample collection Soil samples were collected from the rhizosphere soil of *Pteris vittata* in the tidal flats of the Futian Mangrove Nature Reserve in Shenzhen (144°05′E, 22°51′N). The samples were quickly placed in sterile bags, placed on ice, and transported back to the laboratory for storage at 4°C.

[0044] (2) Resistance to heavy metal Cd 2+ Ni 2+ Cu 2+ and Zn 2+ Culture and isolation of growing microorganisms: The strains were screened using the traditional plate culture method: First, 1.0 g of the soil sample collected in step (1) was weighed, mixed with 9 mL of sterile water, and then serially diluted with sterile water to 10 mL each time. -1 10 -2 10 -3 10 -4 10 -5 10 -6 Six gradients were applied, followed by 100 μL of the diluted solution being spread onto R2A solid medium. Glass beads were added, and the medium was shaken in a cross-shaped motion in a clean bench to ensure even distribution. Colony growth was observed daily. Colonies of different colors and morphologies were isolated using the streak plate method and inoculated into new medium for purification culture, yielding the desired results. Figure 1 The colony diagram shown.

[0045] (3) DNA extraction DNA was extracted from the strains isolated in step (2) using the Tiangen reagent kit. The specific operating steps and precautions were performed in accordance with the instructions.

[0046] (4) PCR amplification Microorganisms were identified based on the 16S rDNA sequence obtained in step (3). The 16S rDNA sequence of the strain was amplified using universal primers 27F (5´-AGAGTTTGATCMTGGCTCAG-3´) and 1492R (5´-TACGGYTACCTTGTTACGACTT-3´). The PCR reaction was carried out in a 20 μL volume containing 1 μL template DNA, 0.5 μL each of forward and reverse primers, 10 μL of Taq-DNA polymerase, and 8 μL of ddH2O. The PCR reaction program was as follows: pre-denaturation at 95℃ for 5 min, followed by 32 cycles, including denaturation at 95℃ for 30 s; annealing at 59℃ for 30 s; extension at 72℃ for 2 min, followed by extension at 72℃ for 5 min, and finally storage at 4℃. PCR products were detected by 1.0% agarose gel electrophoresis and sequenced using an ABI 3730xl automated DNA sequencer. Sequencing results were uploaded to the NCBI nucleotide database and compared with nucleotide sequences in GenBank for homology analysis to identify the strain species. The strain was classified into [species name missing] based on 16S rDNA sequence alignment, ANI value calculated from whole-genome sequencing, and DNA-DNA hybridization value. Aestariibaculum The strain is classified as a genus and named accordingly. Aestuariibaculum sp. JKB11. Aestuariibaculum sp. JKB11's neighbor-joining phylogenetic tree, constructed based on the 16S rDNA gene sequence, is as follows: Figure 2 As shown.

[0047] Example 2 Aestuariibaculum sp. JKB11 vs Cd 2+ Ni 2+ Cu 2+ and Zn 2+ Tolerance test The newly isolated and identified strains Aestuariibaculum sp. JKB11 was activated on R2A solid medium, and then single colonies were picked using the streak plate method and inoculated onto plates containing Cd. 2+ Ni 2+ Cu 2+ Zn 2+ After culturing in R2A solid medium at room temperature for 7 days, the presence or absence of bacterial growth on the solid plates is used to determine the bacterial growth. If bacterial growth occurs on a solid plate at a certain concentration, it indicates that the bacteria can tolerate the environment of that concentration of heavy metals and survive. Aestuariibaculum sp. JKB11 contains the heavy metal Cd. 2+ Ni 2+ Cu 2+ Zn 2+ Grown on solid culture medium, for Cd 2+ Ni2+ Cu 2+ Zn 2+ The tolerability levels were 150 mg / L, 400 mg / L, 0 mg / L, and 0 mg / L, respectively.

[0048] Example 3: Immobilization using corn stalk biochar as a carrier Aestuariibaculum sp JKB11 was used to prepare microbial inoculants. Using corn stalk biochar as a carrier to deliver the target strain Aestuariibaculum sp. JKB11 underwent immobilization treatment. First, corn stalk biochar was ground and passed through a 100-mesh sieve. SG culture media containing 0, 0.2, 0.4, 0.6, 0.8, and 1.0 g / L biochar were prepared respectively. Aestuariibaculum sp. JKB11 was activated on R2A solid medium, and then single colonies were picked and placed in SG liquid medium. The seed culture was incubated at 28°C and 200 rpm. When the seed culture reached the logarithmic growth phase, the bacterial concentration was 1.6 g / L. 10 mL of the seed culture (5% inoculum, v / v) was inoculated into 200 mL of SG medium containing different concentrations of biochar (0, 0.2, 0.4, 0.6, 0.8, and 1 g / L, respectively). After incubation at 28°C and 200 rpm for 36–48 h, bacterial cultures with different biochar concentrations (e.g., ...) were obtained. Figure 3 As shown, from left to right, the concentrations of biochar in the bacterial solution are 0, 0.2, 0.4, 0.6, 0.8, and 1 g / L, respectively. Next, the bacterial solution was centrifuged at 8000 rpm for 10 min to collect the microbial inoculum (denoted as biochar-microbial inoculum), and washed twice with physiological saline to remove residual culture medium.

[0049] Example 4: Immobilization using sodium alginate (SA) and polyvinyl alcohol (PVA) as carriers Aestariibaculum sp. JKB11 was used to make microbial inoculants. Using sodium alginate (SA) and polyvinyl alcohol (PVA) as carriers, the target strain was transported... Aestuariibaculum sp. JKB11 is fixed. First, Aestuariibaculum sp.JKB11 was activated on R2A solid medium, and then a single colony was picked and cultured in SG liquid medium at 28°C and 200 rpm in a shaker. When the seed culture reached the logarithmic phase, the bacterial content in the seed culture was 1.6 g / L. 20 mL of seed culture (5% inoculum, v / v) was inoculated into 400 mL of fresh SG liquid medium and cultured under the same conditions for about 36-48 h. Then, 400 mL of bacterial culture was centrifuged at 8000 rpm for 10 min to collect the bacterial cells, and washed twice with physiological saline (0.9% NaCl) to remove residual culture medium. Finally, the bacterial culture was concentrated to 30 mL with physiological saline.

[0050] Then, prepare a PVA-SA (14% PVA and 1% SA, w / v) mixed solution using 100 mL ddH2O and sterilize it. When cooled to 50℃, add bacterial solutions containing different amounts of bacteria according to the table below and mix thoroughly to obtain PVA-SA microbial agents with different ratios. Finally, use an automatic titration device (peristaltic pump and magnetic stirrer) to uniformly add the PVA-SA microbial agent to a 500 mL mixed solution of saturated boric acid and 5% CaCl2 (solvent is ddH2O, pre-sterilized and stored at 4℃) to form the agent. Control the dropping rate at 0.1-0.3 mL / min. Place the saturated boric acid and 5% CaCl2 mixed solution on a magnetic stirrer and control the speed at 300-400 rpm. After titrating for 10-12 h, obtain small, smooth, uniform, and elastic spherical particles that are pale yellow (blank spheres are milky white). Figure 4 As shown. The obtained microspheres were placed on ice for cross-linking for about 4 hours, and the surface moisture was absorbed with paper towels to obtain dried microsphere granular microbial inoculant (denoted as PVA-SA-microbial inoculant), which was collected and stored in a refrigerator at 4°C.

[0051] Table 1. PVA-SA Microbial Agent Formulation Table

[0052] Example 5 Aestuariibaculum sp. JKB11, Microbial Agents for Cd 2+ Ni 2+ Cu 2+ and Zn 2+ Enrichment capacity test Prepare 25 mL of a solution containing 50 mg / L Cd. 2+ Aqueous solutions of Ni with a concentration of 50 mg / L 2+ Aqueous solutions of Cu with a concentration of 50 mg / L 2+ Aqueous solutions of Zn with a concentration of 50 mg / L 2+ Aqueous solution.

[0053] Will Aestuariibaculum sp. JKB11 was activated on R2A solid medium, and then single colonies were picked and cultured in SG liquid medium at 28°C and 200 rpm in a shaker. When the seed culture reached the logarithmic growth phase, 10 mL of the seed culture (5% inoculum, v / v) was inoculated into 200 mL of fresh SG liquid medium and cultured under the same conditions for approximately 36–48 h. The cells were then collected by centrifugation at 8000 rpm for 10 min and washed twice with high-purity water to remove residual culture medium. 33.07 ± 1.38 mg of the solution was added to each of the pre-prepared fresh solutions containing different heavy metal ions. Aestuariibaculum sp. JKB11 cells were cultured in a shaker at 200 rpm and 28°C for 2 h. The supernatant was then collected by centrifugation, and the concentration of metal ions in the supernatant was determined using inductively coupled plasma optical emission spectrometry (ICP-OES, OPTIMA7000, Perkin Elmer). A blank heavy metal solution without bacterial cells was used as a control. Each experiment was performed in triplicate.

[0054] Add 45.0 mg of biochar-microbial inoculant with different biochar concentrations from Example 3 to each of the pre-prepared fresh solutions containing different heavy metal ions. Add 3.0 g of PVA-SA-microbial inoculant with different raw material ratios from Example 4 to each of the pre-prepared fresh solutions containing different heavy metal ions. Incubate in a shaker at 200 rpm and 35°C for 2 h. Collect the supernatant by centrifugation and determine the concentration of metal ions in the supernatant using inductively coupled plasma optical emission spectrometry (ICP-OES, OPTIMA7000, Perkin Elmer). Use a blank solution without added bacteria as a control. Each experiment was performed in triplicate.

[0055] The removal rate (R) and biosorption capacity (Q) of metal ions are calculated by the following formulas (1) and (2). R=(1-C t / C0)×100%(1) Q=(C0-C t )×V / M(2) In the formula, R represents the removal rate of metal ions (%); Q represents the biosorption capacity of metal ions (mg / g); C t C0 is the final concentration of the solution after enrichment of metal ions (mg / L); C0 is the concentration of the solution before enrichment of metal ions (mg / L); V is the volume of the solution containing heavy metal ions (L); for Aestuariibaculum sp. JKB11 bacteria: M is Aestariibaculum sp.JKB11 dry cell mass (g), for microbial inoculants: M is the dry weight (g) of the cells in the microbial inoculant; the final result is expressed as mean ± standard deviation.

[0056] Aestuariibaculum sp. JKB11, Microbial Agents for Cd 2+ Ni 2+ Cu 2+ and Zn 2+ The enrichment capacity test results are as follows Figure 5-8 As shown, where, Figure 5 In sections (a)-(d), one-way ANOVA was used to analyze the differences between the data. P < 0.05 indicates a significant difference between the data, with a > b > ... > f. Specifically, from Figure 5 As shown in (a)-(d), the bacterial solution containing biochar has a good enrichment effect on heavy metals.

[0057] Figure 6 In (a) and (b), the t-test was used to analyze the differences between the data. "" indicates that there is a significant difference between the data, P < 0.05. Specifically, by Figure 6 From (a) and (b), it can be seen that in a solution containing 50 mg / L of Cd... 2+ Ni 2+ Cu 2+ and Zn 2+ In solution, biochar-microbial inoculant (prepared from a bacterial solution of 0.4 g / L biochar) has an effect on the metal ion Cd. 2+ Ni 2+ Cu 2+ and Zn 2+ The removal rates were 36.07%, 22.58%, 37.62%, and 25.62%, respectively, and the adsorption capacities were 10.49 mg / g, 6.65 mg / g, 10.96 mg / g, and 7.90 mg / g, respectively. Aestuariibaculum sp. The removal rates of metal ions by JKB11 (i.e., free bacterial cells) were 22.02%, 14.09%, 32.30%, and 18.90%, respectively, with adsorption capacities of 8.43 mg / g, 5.86 mg / g, 11.50 mg / g, and 7.67 mg / g, respectively. This indicates that biochar-microbial inoculant has a better metal ion removal effect than unfixed strains, and at the optimal biochar ratio (0.4 g / L biochar), it effectively removed 50 mg / L of Cd. 2+ Ni 2+ Cu 2+ and Zn 2+ The removal rate of heavy metal ions in the solution was significantly higher than that of unfixed strains.

[0058] Figure 7 In (a)-(d), one-way ANOVA was used to analyze the differences between the data. P < 0.05 indicates a significant difference between the data, a > b > ... > f. Specifically, by Figure 7 From (a) to (d), we can know that: The removal capacity of PVA-SA microorganisms for heavy metals initially increases and then decreases with increasing bacterial count. This suggests that within a certain bacterial count range, the removal capacity of PVA-SA microorganisms increases with increasing bacterial count, but beyond a certain threshold, the microsphere structure of the PVA-SA microorganisms becomes unstable and difficult to form, thus reducing the removal capacity. The optimal ratio (14g polyvinyl alcohol, 1g sodium alginate, 288mg...) is also relevant. Aestuariibaculum sp. JKB11 bacterial cells, 130 mL of high-purity water, under conditions containing 50 mg / L Cd 2+ Ni 2+ Cu 2+ and Zn 2+ In the solution, the removal rates of metal ions by PVA-SA-microbial bacteria were 49.80%, 56.60%, 73.47%, and 58.01%, respectively, and the adsorption capacities reached 30.54 mg / g, 23.94 mg / g, 27.01 mg / g, and 23.52 mg / g, respectively.

[0059] Figure 8 In (a) and (b), t-tests were used to analyze the differences between the data. "" indicates that there is a significant difference between the data, P < 0.05. Specifically, by Figure 8 From (a) and (b), we can see that: In the optimal ratio (14g polyvinyl alcohol, 1g sodium alginate, 288mg...), Aestuariibaculum sp. JKB11 bacterial cells, 130 mL of high-purity water, containing 50 mg / L of Cd 2+ Ni 2+ Cu 2+ and Zn 2+ In solution, PVA-SA microbial agent has an effect on metal ions Cd. 2+ Ni 2+ Cu 2+ and Zn 2+ The removal rates were 49.80%, 56.60%, 73.47%, and 58.01%, respectively, and the adsorption capacities were 30.54 mg / g, 23.94 mg / g, 27.01 mg / g, and 23.52 mg / g, respectively. (Bacteria) Aestuariibaculum sp.The removal rates of metal ions by JKB11 (i.e., free bacterial cells) were 22.02%, 14.09%, 32.30%, and 18.90%, respectively, with adsorption capacities of 8.43 mg / g, 5.86 mg / g, 11.50 mg / g, and 7.67 mg / g, respectively. This indicates that the removal rates of heavy metal Cd by the immobilized strain using sodium alginate and polyvinyl alcohol as carriers to form gel microspheres are significantly higher than those by the unimmobilized strain. 2+ Ni 2+ Cu 2+ and Zn 2+ It has a higher removal capacity.

[0060] In summary, this invention provides bacteria capable of accumulating heavy metals, their preparation methods, and applications. Aestuariibaculum sp. JKB11 can effectively enrich Cd in solution. 2+ Ni 2+ Cu 2+ and Zn 2+ This shows Aestuariibaculum sp. JKB11 has potential remediation effects in environments contaminated with cadmium, nickel, copper, and zinc. The bacteria provided by this invention are characterized by low cost, low energy consumption, simple operation, recyclability, environmental friendliness, high efficiency, and low likelihood of secondary pollution when treating heavy metal ions, thus facilitating the removal of Cd from polluted water. 2+ Ni 2+ Cu 2+ and Zn 2+ It offers greater possibilities and operability. Meanwhile, Aestuariibaculum sp. JKB11 can produce a large amount of microbial biomass through short-term fermentation. This invention utilizes immobilized bacteria... Aestuariibaculum sp. The microbial agent prepared using JKB11 has achieved its producibility for practical applications. This invention aims to study the utilization of microorganisms in environments contaminated with cadmium, nickel, copper, and zinc. Aestuariibaculum sp. JKB11 lays the foundation for remediation, enriches the microbial library for using microorganisms to remediate heavy metal pollution, and provides technical support for the ultimate realization of using microorganisms to remediate heavy metal pollution in water and soil.

[0061] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A bacterium capable of accumulating heavy metals, characterized in that, The bacteria are classified as... Aestuariibaculum sp. JKB11, the bacteria described is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 27739.

2. The application of bacteria capable of accumulating heavy metals as described in claim 1, characterized in that, The bacteria capable of accumulating heavy metals are used to accumulate heavy metals, wherein the heavy metals are at least one of cadmium, nickel, copper, and zinc.

3. The application according to claim 2, characterized in that, The bacteria are immobilized to form a microbial agent, which is then used to enrich at least one of cadmium, nickel, copper, and zinc.

4. A microbial inoculant, characterized in that, It includes a carrier and bacteria loaded on the carrier, wherein the bacteria are the heavy metal-accumulating bacteria as described in claim 1.

5. The microbial agent according to claim 4, characterized in that, The carrier is biochar; or, the carrier includes sodium alginate and polyvinyl alcohol.

6. A method for preparing a microbial inoculant, characterized in that, Including the following steps: Bacteria are loaded onto a carrier to obtain the microbial agent; the bacteria are the heavy metal-accumulating bacteria as described in claim 1.

7. The preparation method according to claim 6, characterized in that, The preparation method of the microbial agent specifically includes the following steps; The bacteria capable of accumulating heavy metals were added to a culture medium containing biochar, cultured, and then centrifuged to obtain the microbial inoculum.

8. The preparation method according to claim 6, characterized in that, The preparation method of the microbial agent specifically includes the following steps: providing a mixed solution containing sodium alginate and polyvinyl alcohol; The bacteria capable of accumulating heavy metals are added to the mixed solution to form a microbial inoculant solution; The microbial agent solution is added dropwise to a mixed solution of boric acid and CaCl2 to obtain the microbial agent.

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