Bacillus cereus ZHX-987 and application thereof in synchronously stabilizing soil available cadmium and arsenic

Abstract

Bacillus cereus ZHX-987 and application thereof in synchronously stabilizing soil available cadmium and arsenic, belong to the field of environmental microorganism technology, the bacillus cereus strain ZHX-987 is preserved in China typical culture preservation center, the preservation address is Wuhan University in Wuhan City, Hubei Province, the preservation date is April 27, 2026, and the preservation number is CCTCC NO: M2026831.The above-mentioned strain is oxidized into Fe (III) by biological metabolism, and rapidly forms high-activity secondary iron oxide (iron (hydrogen) oxide secondary mineral). The surface of these new minerals is rich in active functional groups such as iron hydroxyl, which can simultaneously fix cadmium cations (Cd 2+ ) and arsenic anions (arsenite AsO3 3‑ , arsenate AsO4 3‑ ) through adsorption.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, specifically to a Bacillus cereus ZHX-987 and its application in the simultaneous stabilization of available cadmium and arsenic in soil. Background Technology

[0002] Cadmium (Cd) and arsenic (As) are typical heavy metal compound pollutants in farmland soils in my country. Cadmium mainly exists in soil as a divalent cation (Cd). 2+ Arsenic exists primarily as arsenite (AsO3) in the form of arsenite. 3- Arsenate (AsO4) 3- Cadmium and arsenic exist in the form of oxygen-containing anions, and their chemical behaviors and charge properties are significantly different, making it difficult for traditional single passivation materials and repair technologies to achieve simultaneous and efficient fixation of cadmium and arsenic.

[0003] Currently, among the mainstream remediation technologies for cadmium and arsenic co-contaminated soils, physicochemical techniques such as topsoil replacement, leaching, and thermal solidification suffer from high engineering costs, damage to soil aggregate structure, decreased soil fertility, and high risk of secondary pollution, making them difficult to promote and apply in large-scale farmland remediation. Microbial remediation technology has become a research hotspot due to its environmental friendliness, low cost, and suitability for in-situ remediation. However, existing technologies still have significant bottlenecks. Conventional microbial remediation techniques often rely on functionally specific strains; for example, certain Bacillus or Pseudomonas bacteria can adsorb Cd through cell surface functional groups. 2+ However, its ability to fix arsenic is weak; while Thiobacillus or arsenic-oxidizing bacteria can oxidize highly toxic As(III) to less toxic As(V), their removal effect on Cd is limited. It is worth noting that relying solely on valence state transformation cannot achieve long-term stable arsenic fixation, and the risk of secondary activation remains. These limitations generally limit the remediation efficacy of existing technologies for sites contaminated with both cadmium and arsenic.

[0004] In recent years, strategies based on iron-oxidizing bacteria inducing iron mineralization and synergistically immobilizing multiple heavy metals have shown great promise. The iron (hydride) oxide secondary minerals produced by iron-oxidizing bacteria can simultaneously immobilize cadmium cations and arsenic anions through adsorption and co-precipitation. However, this technology still faces significant bottlenecks in practical applications: on the one hand, most known iron-oxidizing bacteria have poor tolerance to high concentrations of cadmium and arsenic combined pollution, and their metabolic activity and iron oxidation capacity are easily inhibited under pollution stress; on the other hand, existing research mainly focuses on the removal of heavy metals from aquatic media, while multifunctional strains with both high iron oxidation efficiency and tolerance to cadmium and arsenic combined pollution are still scarce in soil media, and the corresponding bacterial agent preparation and application technology system is still incomplete.

[0005] Therefore, the urgent technical problem to be solved is to screen out functional strains that have high tolerance to cadmium and arsenic, high iron oxidation activity, and strong adaptability to soil environment, and to establish standardized bacterial agent preparation processes and in-situ remediation methods, so as to simultaneously stabilize and fix available cadmium and arsenic in the soil through adsorption and co-precipitation driven by high iron oxidation. Summary of the Invention

[0006] To address the above problems, this invention provides Bacillus cereus ZHX-987 and its application in the simultaneous stabilization of available cadmium and arsenic in soil, aiming to overcome the shortcomings of existing environmental microbial remediation technologies.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A strain of Bacillus cereus, ZHX-987, is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, Hubei Province, on April 27, 2026, with accession number CCTCC NO: M2026831.

[0009] The aforementioned strains oxidize Fe(II) to Fe(III) through biological metabolism, rapidly forming highly reactive secondary iron oxides (iron (hydride) oxide secondary minerals). These newly formed minerals are rich in active functional groups such as iron hydroxyl groups on their surface, simultaneously immobilizing cadmium cations (Cd) through adsorption. 2+ ) and arsenite anion (arsenite AsO3) 3- Arsenate (AsO4) 3- ).

[0010] A bioremediation agent for cadmium-arsenic co-contaminated soil comprises the aforementioned Bacillus cereus strain ZHX-987 and a freeze-drying protectant, wherein the bioremediation agent has a viable bacterial count of not less than 1.0 × 10⁻⁶. 7 A lyophilized powder formulation with a concentration of CFU / g.

[0011] In a preferred embodiment, the lyophilization protectant comprises 5-10% (w / v) trehalose and 1-5% (w / v) glycerol.

[0012] Trehalose and glycerol in the freeze-drying protectant play a protective role during vacuum freeze-drying. Trehalose forms a protective layer on the surface of the bacterial cell membrane, replacing water molecules in hydrogen bonding with membrane lipids and proteins, preventing mechanical damage to the cell membrane caused by ice crystal formation. Glycerol, as a permeable protectant, can penetrate into the cell interior, lowering the intracellular ice crystal formation temperature and maintaining the native conformation of proteins. The synergistic effect of these two components ensures high survival rates of the bacteria during freeze-drying and storage. After application to soil, the ZHX-987 bacteria in the freeze-dried powder are rehydrated and activated, using naturally occurring or added ferrous salts (such as ferric ammonium citrate) as substrates to produce secondary iron oxides through iron oxidation metabolism. These secondary iron oxides simultaneously fix available cadmium ions and arsenite / arsenate ions in the soil through surface complexation adsorption and co-precipitation.

[0013] A method for preparing a bioremediation microbial agent includes the following steps:

[0014] S1: The Bacillus cereus strain ZHX-987 was activated and cultured, and seed culture with an OD600 value of 1.0-2.0 was prepared by step-by-step expansion.

[0015] S2: The seed culture is inoculated into a fermentation medium containing ferrous salt at a volume ratio of 2%-10% for expanded fermentation culture to obtain a high-density bacterial suspension;

[0016] S3: Mix the high-density bacterial suspension with a freeze-drying protectant until homogeneous, and then freeze-dry under vacuum to obtain a powdered microbial agent.

[0017] In a preferred embodiment, step S1 specifically includes the following process:

[0018] A1: The Bacillus cereus strain ZHX-987 was inoculated into liquid activation medium under aseptic conditions and cultured at 30-35℃ for 24-48h to obtain activated culture;

[0019] A2: Transfer the activated culture to an Erlenmeyer flask containing 50-100 mL of liquid seed culture medium and culture with shaking at 25-30℃ and 100-150 rpm for 12-24 h until the OD600 value of the culture medium is 1.0-2.0 to obtain the primary seed culture.

[0020] A3: The primary seed culture is transferred again to fresh liquid seed culture medium at an inoculation rate of 1%-5% by volume for secondary expansion. The culture conditions are the same as those for preparing the primary seed culture, to obtain a seed culture with an OD600 value of 1.0-2.0.

[0021] In a preferred embodiment, the liquid activated culture medium consists of the following components: 10 g / L trypsin, 5 g / L yeast extract, and 10 g / L NaCl.

[0022] In a preferred embodiment, step S2 specifically includes: inoculating the seed liquid into a fermentation medium containing ferrous salt at a volume ratio of 2%-10%, and culturing it at 30-37℃ and 100-200 rpm for 48-96 h.

[0023] Bacillus cereus ZHX-987 is a facultative aerobe, and its iron oxidation metabolism requires a high level of dissolved oxygen. The culture temperature was within the optimal growth range for this strain, resulting in the highest activity of its metabolic enzymes (including iron oxidation enzymes). Stirring speed functioned through the following mechanisms: ① increasing the gas-liquid contact area and raising the dissolved oxygen concentration in the culture medium; ② promoting the uniform distribution of nutrients and ferrous salts in the culture medium; ③ preventing bacterial cell precipitation and aggregation; ④ promoting the uniform dispersion of metabolites (including secondary iron oxides), avoiding excessively high local concentrations that could cause toxicity to the bacteria. The culture time covered the logarithmic and stationary phases of the bacterial growth curve: the first 48 hours were the stage of rapid bacterial proliferation and iron oxidation induction, while the latter 48 hours were the stage of significant accumulation of iron oxidation products and secondary mineral formation. The optimal harvest time was determined by monitoring OD600 values, pH changes, or residual sugar levels to ensure that both bacterial density and iron oxidation activity were at their best at harvest.

[0024] In a preferred embodiment, the fermentation medium consists of the following components: glucose 2-5 g / L, tryptone 3-8 g / L, yeast extract 1-3 g / L, (NH4)2SO4 0.5-2 g / L, KH2PO4 0.5-1.5 g / L, MgSO4·7H2O 0.2-0.5 g / L, CaCl2 0.05-0.2 g / L, and ferric ammonium citrate 5-20 g / L, with the initial pH of the medium adjusted to 6.5-7.5.

[0025] Ferric ammonium citrate plays a dual role in this culture medium. On the one hand, it acts as an iron source (Fe... 3+ / Fe 2+ Ferrous ammonium citrate provides a substrate for the iron oxidation metabolism of bacteria. On the other hand, the citrate ions in ferric ammonium citrate act as chelating agents for iron ions, keeping them soluble in the culture medium and facilitating uptake and metabolism by bacteria. As a heterotrophic microorganism, *Bacillus cereus* ZHX-987, during its fermentation metabolism using carbon sources such as glucose, utilizes the cell membrane-associated electron transport chain and the enzymatic action of intracellular iron reductase to uptake iron from the cell or its surface. 3+ Reduced to Fe 2+ This reduction process consumes the reducing power generated by metabolism and is a necessary activation step for bacteria to obtain the substrates needed for subsequent oxidative metabolism. The Fe produced by the reduction... 2+ It is then re-oxidized by the ferrooxidase system of the bacteria (under aerobic conditions) into highly active nascent Fe.3+ This oxidation step is an important supplementary step in the energy metabolism of this strain. Its core purpose is not to obtain energy, but to directionally produce highly reactive nascent Fe. 3+ Ions. These newly formed Fe atoms 3+ It exhibits extremely high chemical activity. Under conditions of initial pH 6.5-7.5 (near neutral) in the culture medium, it immediately breaks free from the binding of the chelating agent, rapidly undergoing strong hydrolysis and polymerization to form a large amount of amorphous hydrated iron oxide (iron oxide) secondary mineral precipitate. It is this cascade mechanism of "bioreduction providing substrate → biooxidation driving precipitation" that enables the iron oxidation rate to exceed 80% within 120 hours, and the total iron reduction rate in the liquid phase to approach 100%. The initial pH adjustment of 6.5-7.5 is within the optimal pH range for ferrooxidase and the suitable pH range for cell growth.

[0026] In a preferred embodiment, the mass ratio of the high-density bacterial suspension to the freeze-drying protectant is 1-2:1; the vacuum freeze-drying conditions are as follows: pre-freezing at -80℃ for 3-4 h; then drying for 12-18 h under vacuum of 10-20 Pa and shelf temperature of -25℃ to -15℃; and finally drying for 4-6 h under vacuum of 10-20 Pa and shelf temperature gradually increased from 0℃ to 20℃.

[0027] Application of Bacillus cereus strain ZHX-987 in the simultaneous stabilization of available cadmium and arsenic in soil. The Bacillus cereus strain ZHX-987 was applied as a bioremediation agent. The application rate of the bioremediation agent was such that the viable count of the strain reached 1.0 × 10⁻⁶ bacteria per gram of cadmium- and arsenic-contaminated soil. 7 CFU or higher, for example, when prepared as a solid bacterial powder with a live bacteria content of 1.0 × 10⁻⁶. 8 When the concentration of CFU / g is 0.1g, the application rate is 0.1g per kilogram of soil. Applying the microbial remediation agent to contaminated soil utilizes the highly efficient iron oxidation-driven adsorption and co-precipitation of Bacillus cereus strain ZHX-987 to simultaneously remove available cadmium and arsenic from the soil, thereby achieving long-term immobilization of cadmium and arsenic.

[0028] After the bioremediation agent was applied to the soil, strain ZHX-987 utilized naturally occurring or added ferrous salts (such as ferric ammonium citrate) in the soil as substrates to oxidize Fe(II) to Fe(III) through its iron oxidation metabolic pathway. Under near-neutral to slightly acidic soil pH conditions (pH 5.99 of the tested soil), Fe(III) rapidly hydrolyzed and polymerized to form highly reactive secondary iron (hydride) oxides (such as goethite and ferrihydrite). These newly formed iron oxides possess a large specific surface area and abundant surface active sites.

[0029] In soils contaminated with cadmium and arsenic, cadmium mainly exists as a divalent cation (Cd). 2+ Arsenic exists primarily as arsenite (AsO3). 3- ) and arsenate (AsO4) 3- It exists in the form of oxygen-containing anions, such as α-oxoanions. The ferric hydroxyl groups on the surface of newly formed iron oxides can simultaneously adsorb positively charged Cd through ligand exchange reactions. 2+ And negatively charged arsenic oxyacid ions. For Cd 2+ FeOCd is mainly formed through surface complexation reactions. + Or inner-layer complexes such as FeOCdOH; for arsenate / arsenite, mainly through ligand exchange to form bidentate complexes such as FeOAsO3H or FeOAsO2H. Furthermore, co-precipitation during iron oxide formation can directly encapsulate some Cd and As into the mineral lattice, achieving more stable fixation. Through the synergistic effect of the above adsorption and co-precipitation, bioavailable exchangeable and carbonate-bound cadmium and arsenic in the soil are converted into stable iron oxide-bound and residual states, thus simultaneously achieving long-term stabilization and fixation of cadmium and arsenic.

[0030] Compared with the prior art, the technical advantages of the present invention are:

[0031] (1) In view of the shortcomings of existing soil remediation technologies for cadmium and arsenic co-contaminated soil, such as single function, difficulty in simultaneously passivating cadmium and arsenic, poor long-term stability, and insufficient environmental adaptability, the bioremediation method proposed in this invention targets cadmium and arsenic co-contaminated soil. By applying a specific target microbial agent, namely Bacillus cereus strain ZHX-987, the effective iron oxidation-driven adsorption and co-precipitation effect of the strain is used to simultaneously remediate available cadmium and arsenic in the soil, thereby achieving long-term stable fixation of cadmium and arsenic. This strain was isolated from a cadmium and arsenic co-contaminated farmland soil sample in Liuyang City, Changsha City, Hunan Province. It was obtained through cadmium and arsenic concentration gradient pressure screening and showed strong tolerance to cadmium and arsenic. In particular, it can still maintain iron oxidation activity and cadmium and arsenic removal efficiency under conditions where the arsenic concentration is as high as 10 mg / L or more. The removal rate of available cadmium and arsenic can reach more than 80%. At the same time, a matching high-density agent preparation process is established to ensure the colonization ability and remediation efficiency of the strain in the contaminated soil. This strain, as an excellent iron-oxidizing microorganism, has advantages such as no risk of secondary pollution, high treatment efficiency, and good environmental adaptability when applied to the bioremediation of farmland soil contaminated with cadmium and arsenic.

[0032] (2) By optimizing the type and concentration ratio of the freeze-drying protectant, efficient protection of Bacillus cereus ZHX-987 cells was achieved during vacuum freeze-drying and storage. Trehalose and glycerol work synergistically, with the former focusing on protecting the natural structure of the cell membrane and proteins, and the latter focusing on reducing the risk of intracellular ice crystal damage. Using this freeze-drying protectant, the viable cell survival rate of the bacterial agent can be higher than 60% after 6 months of storage at room temperature, in a dry and dark environment, which significantly extends the shelf life of the bacterial agent and reduces storage and transportation costs.

[0033] (3) Through the complete process of “activation culture → step-by-step expansion → seed liquid preparation → expansion culture in ferrous salt-containing fermentation medium → lyophilization protectant mixing → vacuum freeze-drying”, the standardized and large-scale production of ZHX-987 strain from its preservation state to high-activity powdered bacterial agent was realized. This method is relatively simple and cost-controllable, and can stably produce efficient and highly active functional microbial agents. By adding ferrous salt (ferric ammonium citrate) to the fermentation medium, the iron oxidation metabolic activity of the strain is simultaneously induced during the expansion culture process, so that the final bacterial agent product not only contains high-density live bacteria, but also maintains the iron oxidation functional characteristics of the strain. The vacuum freeze-drying step makes the product exist in powder form with an effective live bacteria count of not less than 1.0 × 10⁻⁶. 7 CFU / g, which facilitates long-term storage and field transportation and application. Attached Figure Description

[0034] Figure 1 This is a typical colony morphology diagram of strain ZHX-987 in Example 1 on a screening plate;

[0035] Figure 2 This is the ZHX-987 phylogenetic tree constructed based on the 16S rRNA sequence in Example 1;

[0036] Figure 3 This is a growth curve of strain ZHX-987 in Example 3 under single or combined cadmium (Cd) / arsenic (As) stress;

[0037] Figure 4 Figure 1 shows the results of the iron oxidation activity and cadmium and arsenic removal capacity test of strain ZHX-987 in Example 3. (a) shows the cadmium removal curve of strain ZHX-987 under different heavy metal treatments, (b) shows the arsenic removal curve of strain ZHX-987 under different heavy metal treatments, (c) shows the Fe(II) oxidation rate curve of strain ZHX-987 under different heavy metal treatments, and (d) shows the total iron (TFe) reduction rate curve of strain ZHX-987 under different heavy metal treatments.

[0038] Figure 5 This is an experimental diagram showing the formation of iron oxide precipitate by iron oxidation reaction of strain ZHX-987 in Example 3;

[0039] Figure 6 This is a photograph of the application of Bacillus cereus strain ZHX-987 in soil contaminated with cadmium and arsenic. Detailed Implementation

[0040] To enable those skilled in the art to better understand the technical solution, the present invention will be described in detail below with reference to embodiments. The description in this part is only exemplary and explanatory, and should not be used to limit the scope of protection of the present invention in any way.

[0041] The following examples use Bacillus cereus ZHX-987, which was deposited on April 27, 2026, at the China Center for Type Culture Collection (CCTCC, located at Wuhan University, Wuhan, Hubei Province), with accession number CCTCC NO: M2026831.

[0042] Example 1:

[0043] Isolation, identification and preservation of Bacillus cereus strain ZHX-987.

[0044] 1) Isolation and purification of Bacillus cereus strain ZHX-987:

[0045] In this embodiment, strain ZHX-987 was isolated from a soil sample of farmland contaminated with cadmium and arsenic in Liuyang City, Changsha City, Hunan Province. 5 g of soil sample was taken, prepared into a soil suspension, and after settling, the supernatant was collected for 10... -5 -10 -8 Gradient dilution.

[0046] The diluted bacterial suspension was inoculated into selective culture medium and cultured with shaking at 28°C and 150 rpm. Subculture was performed every 3-5 days at a 10% inoculum, for a total of 3-5 subcultures. During the process, the concentrations of cadmium (Cd) and arsenic (As) were gradually increased to implement pressure screening.

[0047] Dilute the last culture medium by 10. -2 -10 -8 After dilution, the culture was spread onto solid culture plates and incubated upside down. Once single colonies appeared on the plates, they were picked and purified by repeated streak plating to obtain a pure strain, which was named strain ZHX-987.

[0048] During the isolation and culture of *Bacillus cereus*, the selected liquid culture medium consisted of: 0.5 g / L peptone, 0.3 g / L glucose, 0.2 g / L yeast extract, 0.1 g / L K₂HPO₄, 0.2 g / L MgSO₄·7H₂O, 0.2 g / L NaNO₃, 0.1 g / L CaCl₂, 0.1 g / L (NH₄)₂CO₃, and 2 mL / L ferric citrate solution (2.83 g ferrous sulfate and 3 g trisodium citrate were dissolved separately, mixed, and brought to a final volume of 20 mL, filtered through a 0.22 μm sterile membrane, and then frozen). The specific preparation process was as follows: the components of the above liquid culture medium were dissolved in water and sterilized at 121°C for 20 min. The selected solid culture plates were prepared by adding 2% (w / v) agar to the liquid culture medium.

[0049] 2) Morphological identification of Bacillus cereus strain ZHX-987

[0050] The ZHX-987 strain was inoculated onto solid selective medium using the streak plating method, and colony morphology was observed. Results are as follows: Figure 1 The colony was found to be approximately 1 mm in diameter, round, brownish-red, with a rough, waxy surface and irregular edges.

[0051] 3) Molecular biological identification of Bacillus cereus strain ZHX-987

[0052] Total DNA was extracted from the strain using SDS or STE methods. The obtained DNA was diluted to approximately 50 ng / μL and used as a template for amplification of the 16S rDNA gene. Universal primers (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used for PCR amplification of the strain's 16S rDNA gene. After sequencing, the amplified products were submitted to the GenBank database (accession number SUB16192692) for homology comparison. Online homology comparison with sequences in the GenBank database preliminarily identified that strain ZHX-987 shared 99% homology with *Bacillus*. Furthermore, phylogenetic trees were constructed using closely related species (e.g.,...). Figure 2 As shown in the figure, based on the analysis of phylogenetic relationships, it is speculated that this bacterium belongs to a new strain of Bacillus.

[0053] The strain was named Bacillus cereus ZHX-987 and was deposited at the China Center for Type Culture Collection (CCTCC) on April 27, 2026, with accession number CCTCC NO: M2026831.

[0054] Example 2:

[0055] This embodiment prepares a powdered remedial agent of Bacillus cereus strain ZHX-987. The preparation method includes the following steps:

[0056] Step 1: Strain activation and seed culture preparation

[0057] The ZHX-987 strain, preserved in glycerol tubes or slant at -80℃, was aseptically transferred to an inoculation liquid activation medium (10 g / L trypsin, 5 g / L yeast extract, 10 g / L sodium chloride). The culture was incubated at 30℃ with static or shaking (100 rpm) for 48 h to obtain an activated culture. An appropriate amount of the activated culture was transferred to an Erlenmeyer flask containing 100 mL of liquid seed medium and incubated with shaking at 30℃ and 100 rpm for 24 h until the OD600 value was between 1.0 and 2.0, thus obtaining a highly active primary seed culture. To obtain a more uniform and viable inoculum, the primary seed culture was transferred again to fresh seed medium at an inoculation rate of 5% (v / v) for secondary expansion under the same conditions to prepare the seed culture for fermentation inoculation.

[0058] Step 2: Fermentation scale-up culture

[0059] The seed culture prepared in step 1 was aseptically inoculated at a rate of 10% (v / v) into a fermenter or large Erlenmeyer flask containing a ferrous salt fermentation medium. The fermentation medium formulation included: carbon source (e.g., glucose 5 g / L), nitrogen source (e.g., tryptone 8 g / L, yeast extract 3 g / L, (NH4)2SO4 2 g / L), inorganic salts (KH2PO4 1.5 g / L, MgSO4·7H2O 0.5 g / L, CaCl2 0.2 g / L), and ferric ammonium citrate 10 g / L. The initial pH of the medium was adjusted to 7.5. Fermentation was carried out under controlled temperature, aeration, and stirring conditions. Specifically, the culture temperature was controlled at 37℃, the stirring speed was set to 200 rpm according to the container size to ensure sufficient dissolved oxygen, and the culture time was 60 h. The optimal harvest time was determined by monitoring the OD600 value, pH, or residual sugar content. Meanwhile, during the fermentation process, carbon sources (such as glucose) and iron salts can be added in batches through a feed-feeding method to further improve the final cell density and iron oxidase activity.

[0060] Step 3: Collection of bacterial cells and preparation of inoculum

[0061] After fermentation, the high-density bacterial suspension obtained is used to collect wet bacterial cells by centrifugation or membrane filtration. The obtained wet bacterial cells are then thoroughly mixed with a composite freeze-drying protectant under aseptic conditions at low temperature (4°C) and resuspended to prepare a bacterial suspension. The composite freeze-drying protectant contains nutrient protectants (such as 8% (w / v) trehalose and 5% (w / v) glycerol) and pH buffer salts, with a total mass fraction of 15% for nutrients and buffer salts in the protectant solution. The uniformly mixed bacterial suspension is pre-frozen at -80°C for 4 hours, and then placed in a freeze dryer for a first drying of 15 hours under a vacuum of 10-20 Pa and a shelf temperature of -25°C to -15°C. A second drying is then performed for 6 hours under a vacuum of 10 Pa and a shelf temperature gradually increased from 0°C to 20°C, until the moisture content of the material is below 5%. The dried lumpy material is then pulverized and sieved at low temperature to obtain a highly active pure bacterial freeze-dried powder preparation. The effective viable count of this preparation is not less than 1.0 × 10⁻⁶. 7 After being stored at room temperature, in a dry environment, and protected from light for 6 months, the survival rate of live bacteria is higher than 60% (CFU / g). Before application, this dry powder bacterial agent can be directly reconstituted in sterile water to prepare a high-concentration bacterial suspension.

[0062] Example 3:

[0063] Cadmium and arsenic tolerance and functional performance testing of Bacillus cereus strain ZHX-987.

[0064] 1) Cadmium and arsenic tolerance test of strain ZHX-987:

[0065] This embodiment tested the tolerance of the above-mentioned Bacillus cereus strain to cadmium and arsenic. Freshly activated Bacillus cereus seed culture ZHX-987 was inoculated at a rate of 5% (v / v) into Erlenmeyer flasks containing 100 mL of liquid culture medium (composition as above). Four treatment groups were set up using CdCl2 and Na2HAsO4·7H2O as contaminants: ① Blank control group (CK, no cadmium or arsenic added, no inoculation); ② Cd single contamination group (Cd concentration 1 mg / L); ③ As single contamination group (As concentration 10 mg / L); ④ Cd+As combined contamination group (Cd 1 mg / L + As 10 mg / L). Each inoculation group had three replicates.

[0066] The treatment group was cultured at 30℃ and 150 rpm with shaking. At 0h, 4h, 8h, 12h, 16h, 20h, 24h, 36h, 48h, 72h, and 96h after culture, 5 mL samples were aseptically taken to directly measure the optical density (OD600) of the bacterial solution at 600 nm wavelength, so as to quantitatively reflect the growth of bacteria under different cadmium and arsenic stress concentrations.

[0067] The results are as follows Figure 3The results showed that *Bacillus cereus* maintained normal growth and proliferation under single heavy metal stress conditions, namely cadmium concentration of 1 mg / L or arsenic concentration of 10 mg / L. Although its growth curve was slightly lower than the control group without heavy metal addition throughout the culture period, it still exhibited a clear logarithmic growth phase and plateau phase, indicating that the strain was not significantly inhibited under these two single stress conditions. However, in the combined stress treatment group of cadmium (1 mg / L) and arsenic (10 mg / L), the growth curve of the strain decreased significantly, and the OD values ​​and final cell density at each time point were lower than those in the single stress group, but not significantly. This indicates that the strain has excellent tolerance to cadmium and arsenic, and this tolerance is partially maintained under combined pollution conditions. This characteristic is an important biological basis for ensuring the successful colonization and remediation function of the strain in a cadmium-arsenic combined pollution environment.

[0068] 2) Tests on iron oxidation activity and cadmium and arsenic removal capacity of strain ZHX-987

[0069] This embodiment uses the same cadmium and arsenic tolerance settings as described above. Under the same culture conditions, the iron oxidation activity of strain ZHX-987 and its removal capacity for cadmium and arsenic were further determined. The results showed that in both the single Cd (1 mg / L) and Cd+As composite systems, the Cd(II) concentration plateaued during the first 48 hours of reaction, then rapidly decreased, reaching below 0.2 mg / L in the liquid phase by 120 hours (e.g., ...). Figure 4 (a) shows the concentration changes of As(III). Similarly, the concentration changes of As(III) also show obvious phased characteristics (e.g. Figure 4 (b) shows that the As(III) concentration remained at a high level from 0 to 48 h, but decreased sharply from 48 to 120 h, eventually dropping to a residual concentration of approximately 2 mg / L. Furthermore, ZHX-987 maintained highly efficient iron oxidation activity in the coexistence of Cd(II) and As(III) (e.g., Figure 4 (c) shows that after a functional induction period of approximately 48 h, the Fe(II) oxidation rate entered a rapid growth phase, exceeding 80% in all inoculation treatment groups by 120 h, significantly better than the abiotic oxidation level of the control group (CK) (approximately 30%). Correspondingly, the distribution of total iron (TFe) in the liquid phase showed an evolution trend highly synchronized with iron oxidation (e.g., Figure 4 (d) As the reaction proceeded, the reduction rate of TFe in the liquid phase (i.e., the proportion converted to the solid phase) increased synchronously with the increase of Fe(II) oxidation rate. At the end of the 120 h reaction, the reduction rate of TFe in the liquid phase of each inoculated group was close to 100%, while the control group showed almost no change. The results are as follows... Figure 5As shown, this indicates that the strain possesses iron oxidation capabilities, generating iron oxide precipitates. These results demonstrate that ZHX-987 oxidizes Fe(II) to Fe(III) through biological metabolism, rapidly forming highly active secondary iron oxides. These newly formed minerals achieve effective fixation of Cd(II) and As(III) in the aqueous phase through efficient adsorption or co-precipitation.

[0070] Example 4:

[0071] Changes in the bioavailable content of cadmium and arsenic in paddy soil contaminated with cadmium and arsenic by strain ZHX-987.

[0072] The tested soil samples were collected from typical paddy fields in southern rice-growing areas, with a sampling depth of 0-20 cm in the topsoil layer. A five-point mixed sampling method was used, with five sampling points evenly selected within the sampling area. After removing stones, plant debris, and other impurities, the collected soil samples were placed in a well-ventilated, shady place to air-dry naturally for 7-10 days until constant weight. The samples were then sieved through a 2mm nylon sieve, and unbroken gravel, plant roots, and other impurities were manually removed. The samples were thoroughly mixed before use. The initial basic physicochemical properties of the tested soil were: pH 5.99, Cd content 0.804 mg / kg, As content 30.6 mg / kg, available Cd 0.36 mg / kg, and available As 6.81 mg / kg. 350 g of the pretreated, air-dried soil was placed in each 500 mL polyethylene culture dish, spread evenly to avoid soil stratification. Then, 200 mL of ultrapure water and ferric ammonium citrate solution were added, mixed thoroughly, and left to stand for 7 days to reduce the soil moisture content, eventually maintaining a soil moisture content of 30%. Weigh 1.0 g of the lyophilized bacterial powder prepared in Example 2 (viable count 1×10⁻⁶). 7 The bacterial suspension (CFU / g) was resuspended in sterile water and mixed thoroughly to prepare an OD600 = 1.2. Treatment groups were set up, and the bacterial suspension was evenly sprayed onto the soil surface. Then, using sterile tools, it was thoroughly mixed to ensure uniform contact between the bacterial agent and the 0-15 cm soil layer (e.g., CFU / g). Figure 6 (As shown in the figure). A control group was set up, sprayed with an equal amount of sterile water, and other treatments were the same. The pots were placed in a controlled greenhouse for cultivation, and the temperature was maintained at 25℃-30℃ during the cultivation period. The soil moisture was maintained at 60%-70% of field capacity by regular weighing and watering.

[0073] Sampling was conducted on day 60 post-treatment. Before sampling, the soil in the culture pots was gently stirred with a sterile glass rod. A five-point diagonal sampling method was used, with five sampling points evenly selected in each culture pot. After mixing, composite samples were obtained to ensure spatial representativeness. One portion was immediately used to determine the Cd and As concentrations in the soil pore water (measured by ICP-MS after centrifugation and filtration). The other portion was air-dried, ground, and then analyzed using the Tessier five-step continuous extraction method to systematically analyze the chemical speciation of Cd and As in the soil, focusing on changes in the content of available forms (i.e., exchangeable and carbonate-bound forms).

[0074] The results are shown in Table 1. This *Bacillus cereus* strain exhibited significant removal capabilities for both cadmium and arsenic. Regarding cadmium remediation, the available cadmium content in the treated soil decreased from 0.36 mg / kg in the control group to 0.1 mg / kg, achieving a removal rate of 72.22%; the total cadmium content decreased from 0.8 mg / kg in the control group to 0.19 mg / kg, achieving a removal rate of 76.25%. This indicates that the strain possesses extremely strong cadmium removal capabilities. Regarding arsenic remediation, the available arsenic content in the treated soil decreased from 6.81 mg / kg in the control group to 0.72 mg / kg, achieving a removal rate of 89.42%; the total arsenic content decreased from 30.6 mg / kg in the control group to 4.85 mg / kg, achieving a removal rate as high as 84.15%. These results demonstrate that this *Bacillus* strain exhibits excellent simultaneous remediation performance in soils contaminated with both cadmium and arsenic.

[0075] Table 1. Remediation effects of Bacillus cereus on cadmium (Cd) and arsenic (As) in soil.

[0076]

[0077] It should be noted that, in this document, the terms "comprising," "including," and any other variations are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Specific examples have been used in this document to illustrate the principles and implementation methods of the present invention. These examples are merely for the purpose of helping to understand the method and core ideas of the present invention. The above descriptions are only preferred embodiments of the present invention. It should be pointed out that, due to the limitations of written expression and the objective existence of infinite specific structures, those skilled in the art can make several improvements, modifications, or variations without departing from the principles of the present invention, and can also combine the above technical features in an appropriate manner. These improvements, modifications, variations, or combinations, or the direct application of the concept and technical solution of the present invention to other situations without modification, should all be considered within the scope of protection of the present invention.

Claims

1. A Bacillus cereus strain ZHX-987, characterized in that, The Bacillus cereus strain ZHX-987 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026831.

2. A bioremediation agent for cadmium-arsenic co-contaminated soil, characterized in that, The product comprises the Bacillus cereus strain ZHX-987 as described in claim 1 and a lyophilization protectant, wherein the bioremediation agent has a viable count of not less than 1.0 × 10⁻⁶ cells / year. 7 A lyophilized powder formulation with a concentration of CFU / g.

3. The bioremediation agent according to claim 2, characterized in that, The freeze-drying protectant contains 5-10% (w / v) trehalose and 1-5% (w / v) glycerol.

4. A method for preparing the bioremediation agent according to any one of claims 2-3, characterized in that, Includes the following steps: S1: The Bacillus cereus strain ZHX-987 was activated and cultured, and seed culture with an OD600 value of 1.0-2.0 was prepared by step-by-step expansion. S2: The seed culture is inoculated into a fermentation medium containing ferrous salt at a volume ratio of 2%-10% for expanded fermentation culture to obtain a high-density bacterial suspension; S3: Mix the high-density bacterial suspension with a freeze-drying protectant until homogeneous, and then freeze-dry under vacuum to obtain a powdered microbial agent.

5. The preparation method according to claim 4, characterized in that, The specific process of step S1 includes: A1: The Bacillus cereus strain ZHX-987 was inoculated into liquid activation medium under aseptic conditions and cultured at 30-35℃ for 24-48h to obtain activated culture; A2: Transfer the activated culture to an Erlenmeyer flask containing 50-100 mL of liquid seed culture medium and culture with shaking at 25-30℃ and 100-150 rpm for 12-24 h until the OD600 value of the culture medium is 1.0-2.0 to obtain the primary seed culture. A3: The primary seed culture is transferred again to fresh liquid seed culture medium at an inoculation rate of 1%-5% by volume for secondary expansion. The culture conditions are the same as those for preparing the primary seed culture, to obtain a seed culture with an OD600 value of 1.0-2.

0.

6. The preparation method according to claim 5, characterized in that, The liquid activation culture medium consists of the following components: 10 g / L trypsin, 5 g / L yeast extract, and 10 g / L NaCl.

7. The preparation method according to claim 4, characterized in that, The specific process of step S2 includes: inoculating the seed liquid into a fermentation medium containing ferrous salt at a volume ratio of 2%-10%, and culturing it at 30-37℃ and 100-200 rpm for 48-96 h.

8. The preparation method according to claim 7, characterized in that, The fermentation medium consists of the following components: glucose 2-5 g / L, tryptone 3-8 g / L, yeast extract 1-3 g / L, (NH4)2SO4 0.5-2 g / L, KH2PO4 0.5-1.5 g / L, MgSO4·7H2O 0.2-0.5 g / L, CaCl2 0.05-0.2 g / L, and ferric ammonium citrate 5-20 g / L. The initial pH of the medium is adjusted to 6.5-7.

5.

9. The preparation method according to claim 4, characterized in that, The mass ratio of the high-density bacterial suspension to the freeze-drying protectant is 1-2:1; the conditions for vacuum freeze-drying are as follows: pre-freezing at -80℃ for 3-4 h; then drying for 12-18 h under vacuum of 10-20 Pa and shelf temperature of -25℃ to -15℃; and finally drying for 4-6 h under vacuum of 10-20 Pa and shelf temperature gradually increased from 0℃ to 20℃.

10. The application of Bacillus cereus ZHX-987 as described in claim 1 in the simultaneous stabilization of available cadmium and arsenic in soil, characterized in that, The Bacillus cereus strain ZHX-987 was applied as a bioremediation agent, and the dosage of the bioremediation agent was such that the viable count of the strain reached 1.0 × 10⁻⁶ per gram of cadmium-arsenic co-contaminated soil. 7 CFU or above.

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