A method for bioremediation of cadmium-arsenic combined contaminated soil

Abstract

The application discloses a biological remediation method for cadmium-arsenic composite contaminated soil and relates to the technical field of environmental microbial remediation. The biological remediation method comprises the following steps: applying a target microbial agent to contaminated soil, synchronously removing effective cadmium and arsenic in the soil through the biological mineralization of an achromobacter strain LMG 5911, and thereby realizing long-acting immobilization of cadmium and arsenic; the application amount of the target microbial agent is such that the viable bacterial count of the achromobacter strain LMG 5911 in each kilogram of soil reaches 1x10 6 CFU to 1x10 8 CFU. The biological remediation method disclosed by the application is aimed at cadmium-arsenic composite contaminated soil, and through the application of a specific target microbial agent, namely the achromobacter strain LMG 5911, the biological mineralization of the achromobacter strain LMG 5911 is utilized to synchronously remove effective cadmium and arsenic in the soil, thereby realizing long-term stable immobilization of cadmium and arsenic.

Description

Technical Field

[0001] This application relates to the field of environmental microbial remediation technology, specifically to a bioremediation method for soil contaminated with cadmium and arsenic. Background Technology

[0002] Cadmium (Cd) and arsenic (As) are typical heavy metal compound pollutants in farmland soils in my country, mainly due to their cation composition (Cd, As, and As). 2+ ) and oxygen-containing anions (such as AsO3) 3- The different chemical behaviors of these organisms make it difficult for traditional single chemical passivation techniques to achieve simultaneous and efficient fixation, resulting in significant challenges in their treatment.

[0003] Current remediation technologies, such as topsoil replacement and solidification stabilization, often lead to soil structure damage and are costly, limiting their widespread application in large-scale farmland remediation. Conventional microbial remediation technologies largely rely on functionally specific bacterial strains; for example, certain Bacillus or Pseudomonas bacteria can adsorb Cd through their 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 to induce iron mineralization and synergistically fix multiple heavy metals have shown great promise. 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 capacity 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, it is urgent to screen out microbial strains that can adapt to soil environments contaminated with cadmium and arsenic, and that possess both high-efficiency iron oxidation capabilities and tolerance to multiple heavy metals, and to develop corresponding application processes, in order to achieve simultaneous, long-term, and stable solidification of cadmium and arsenic through biomineralization. Summary of the Invention

[0006] The main objective of this application is to provide a bioremediation method for soil contaminated with cadmium and arsenic, aiming to address the shortcomings of existing environmental microbial remediation technologies.

[0007] To achieve the above objectives, the technical solutions adopted in the embodiments of this application are as follows: In a first aspect, embodiments of this application provide a bioremediation method for cadmium-arsenic co-contaminated soil, comprising the following steps: The target microbial agent was applied to the contaminated soil, and the biomineralization of Achromobacterium strain LMG 5911 was used to remove available cadmium and arsenic from the soil simultaneously, so as to achieve long-term immobilization of cadmium and arsenic. When the viable count of the bacterial agent is ≥1.0×10⁻⁶ 10 When the concentration of CFU / g is 0.0001 g / kg to 0.1 g / kg soil, the application rate of the microbial agent is 0.0001 g / kg to 0.1 g / kg soil.

[0008] As some optional embodiments of this application, the Achromobacter sp. LMG 5911 has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, Hubei Province, on December 31, 2025, with accession number CCTCC NO: M 20253065, and its identification characteristic is Achromobacter sp. LMG-5911.

[0009] As some optional embodiments of this application, the target microbial agent is prepared by the following steps: The Achromobacterium strain LMG 5911 was activated and cultured to prepare a seed culture; The seed culture was inoculated into a fermentation medium containing ferrous salts for expanded fermentation culture to obtain a high-density bacterial suspension; The high-density bacterial suspension was mixed evenly with a freeze-drying protectant and then freeze-dried under vacuum to obtain the microbial agent.

[0010] As some optional embodiments of this application, the fermentation medium is composed of the following components: tryptone 5.0 g / L-10.0 g / L, yeast extract 3.0 g / L-5.0 g / L, sodium chloride 5.0 g / L-10.0 g / L, ferric ammonium citrate 0.5 g / L-5.0 g / L; pH value is 6.5-7.5.

[0011] As some optional embodiments of this application, the freeze-drying protectant includes: trehalose with a w / v of 5%-10% and glycerol with a v / v of 1%-5%.

[0012] As some optional embodiments of this application, the step of activating and culturing the Achromobacterium strain LMG 5911 to prepare seed culture specifically includes: The Achromobacterium strain LMG 5911 was inoculated into liquid activation medium under aseptic conditions and cultured at 30℃-35℃ for 24 h-48 h to obtain activated culture. The activated culture was transferred to an Erlenmeyer flask containing 50 mL-100 mL of liquid seed culture medium and cultured with shaking at 30℃-40℃ and 100 rpm-150 rpm for 12 h-24 h, until the OD of the culture medium was reduced. 600 A value of 1.0-2.0 yields a primary seed solution; The primary seed culture was transferred again to activation medium at an inoculation rate of 1%-5% (v / v) for secondary expansion under the same culture conditions as the primary seed culture preparation, to obtain the seed culture; the OD of the seed culture... 600 The value is 1.5-3.0.

[0013] As some optional embodiments of this application, the activation culture medium consists of the following components: 10 g / L trypsin, 5 g / L yeast extract, and 5 g / L sodium chloride.

[0014] As some optional embodiments of this application, the step of inoculating the seed culture into a fermentation medium containing ferrous salts for expanded fermentation culture to obtain a high-density bacterial suspension includes: The seed culture is aseptically inoculated into a container containing fermentation medium at an inoculation rate of 2%-10% by volume and cultured at a temperature of 30℃-37℃, a stirring speed of 100 rpm-200 rpm, and a culture time of 48 h-96 h.

[0015] As some optional embodiments of this application, the fermentation medium is composed of the following components: glucose 2 g / L-5 g / L, tryptone 3 g / L-8 g / L, yeast extract 1 g / L-3 g / L, (NH4)2SO4 0.5 g / L-2 g / L, KH2PO4 0.5 g / L-1.5 g / L, MgSO4·7H2O 0.2 g / L-0.5 g / L, CaCl2 0.05 g / L-0.2 g / L, and ferric ammonium citrate 5 g / L-20 g / L, with the initial pH of the medium adjusted to 6.5-7.5.

[0016] As some optional embodiments of this application, the mixing volume ratio of the high-density bacterial suspension to the freeze-drying protectant is 1:1 to 2:1; The vacuum freeze-drying process conditions are as follows: the pre-freezing temperature is -40℃ to -80℃, and the pre-freezing time is 2 h to 6 h; the cold trap temperature for vacuum drying is -50℃ to -60℃, the vacuum degree is maintained at 10 Pa to 30 Pa, and the drying time is 24 h to 48 h, until the sample moisture content is less than 5%.

[0017] Compared to existing technologies, the bioremediation method proposed in this application targets cadmium- and arsenic-contaminated soil. By applying a specific target microbial agent—Achromobacterium strain LMG 5911—it utilizes its biomineralization action to simultaneously remove available cadmium and arsenic from the soil, thereby achieving long-term stable immobilization of cadmium and arsenic. This Achromobacterium strain exhibits strong tolerance to cadmium and arsenic, especially maintaining iron oxidation activity and cadmium and arsenic removal efficiency even at arsenic concentrations as high as 100 mg / L, achieving removal rates of 40%–70% for available cadmium and arsenic. This strain can serve as an excellent iron-oxidizing microorganism for the bioremediation of cadmium- and arsenic-contaminated farmland soil, offering advantages such as no risk of secondary pollution, high treatment efficiency, and good environmental adaptability. Attached Figure Description

[0018] Figure 1 A phylogenetic tree of Achromobacterium LMG 5911 was obtained for Example 1 of this application; Figure 2 The growth curves of Achromobacterium tumefaciens LMG 5911 at different cadmium and arsenic concentrations were obtained for Example 1 of this application; Figure 3 Example 1 of this application obtained the ferrous oxidation rate of Achromobacterium LMG 5911 under different cadmium and arsenic stresses; Figure 4 The simultaneous removal kinetics of cadmium and arsenic by Achromobacterium LMG 5911 were obtained for Example 1 of this application. Detailed Implementation

[0019] It should be made clear that the specific implementation examples described herein are for illustrative purposes only and are not intended to limit this application.

[0020] As described above, in order to overcome the shortcomings of the existing technology, this application provides a colorless bacillus ( Achromobacter Achromobacters sp. was deposited on December 31, 2025, at the China Center for Type Culture Collection (CCTCC, located at Wuhan University, Wuhan, Hubei Province, 430072, China), with accession number CCTCC NO: M20253065 and identification characteristic Achromobacters sp.LMG-5911.

[0021] The Achromobacterium strain provided in this application was isolated from a soil sample contaminated with cadmium and arsenic in Liuyang City, Changsha City, Hunan Province. The isolation of this strain was performed using conventional methods for isolating new strains in the field of microbiology.

[0022] Specifically, 5 g of soil sample was taken, prepared into a soil suspension, and after settling, the supernatant was collected and subjected to 10... -6 Up to 10 -8Serial dilutions were performed and inoculated into selective medium, and cultured with shaking at 28°C and 150 rpm. Subculture was carried out every 3-5 days at a 10% inoculum, for a total of 3-5 subcultures, gradually increasing the concentrations of cadmium (Cd) and arsenic (As) during the process to implement pressure screening. The final culture medium was diluted 10... 6 Times –10 8 After multiplying, the culture was spread onto solid culture plates and incubated upside down. After single colonies appeared on the plates, single colonies were picked and purified and isolated by repeated streak streaking. The colonies were approximately 1 mm in diameter, round, brownish-red, and opaque, and were Gram-negative upon staining.

[0023] The selective liquid culture medium used for the isolation and culture of the achromobacterium can be any culture medium conventionally used in the art, and the solid culture plate is prepared by adding an appropriate amount of agar to the liquid culture medium. Preferably, the liquid culture medium contains 0.5 g / L KH₂PO₄·3H₂O, 0.5 g / L NaNO₃, 0.2 g / L CaCl₂·2H₂O, 0.5 g / L MgSO₄·7H₂O, 0.5 g / L (NH₄)₂SO₄, and 10 g / L ferric ammonium citrate. The specific preparation process is as follows: after dissolving the components of the above liquid culture medium in water, sterilize at 121°C for 20 min.

[0024] Molecular biological identification of strain LMG·5911: Total DNA was extracted from this strain using SDS or STE methods. The obtained DNA was diluted to approximately 50 ng / μL and used as a template for 16S rDNA gene amplification. The full-length sequence was amplified by PCR using universal primers 27F and 1492R and then sequenced. The 16S rDNA gene sequence of strain LMG·5911 was submitted to the GenBank database, and online homology comparison with sequences in the GenBank database was performed. Preliminary identification indicated that strain LMG·5911 belongs to the GenBank family. Achromobacter sp. With a similarity of 99%, the phylogenetic tree of strain LMG·5911 is shown below. Figure 1 As shown.

[0025] Secondly, this application tested the tolerance of the aforementioned Achromobacterium strain to cadmium and arsenic. The strain was able to maintain normal growth under cadmium and arsenic contamination conditions, with a minimum tolerable concentration of 50 mg / L for cadmium and a minimum tolerable concentration of 100 mg / L for arsenic. This tolerance has been verified through systematic liquid culture growth experiments.

[0026] Specifically, the tolerance test is performed as follows: Freshly activated Achromobacterium seed culture was inoculated at a rate of 5% (v / v) into Erlenmeyer flasks containing 100 mL of liquid culture medium (composition as above). CdCl2 and Na2HAsO4·7H2O were used as contamination sources. Single and combined contamination treatments were established with cadmium concentrations of 1 mg / L, 10 mg / L, 50 mg / L, and 100 mg / L, and arsenic concentrations of 1 mg / L, 10 mg / L, 50 mg / L, and 100 mg / L. A culture medium containing the corresponding concentrations of cadmium and arsenic was used as a blank control.

[0027] The inoculated culture system was incubated with shaking at 30℃ and 150 rpm. At 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h after incubation, 5 mL samples were aseptically collected for direct measurement of the optical density (OD) of the bacterial culture at 600 nm. 600 The value was used to quantitatively reflect the growth of bacteria under different cadmium and arsenic stress concentrations.

[0028] In treatment groups with cadmium concentrations as high as 50 mg / L and arsenic concentrations as high as 100 mg / L, this strain still exhibited significant growth and proliferation (OD200). 600 The value continued to rise), especially in the treatment of high concentrations of arsenic, its growth curve showed a trend consistent with the uncontaminated or low-contaminated concentration groups, demonstrating its excellent tolerance to high concentrations of arsenic. This characteristic is the biological basis for ensuring that this strain can successfully colonize and perform remediation functions in cadmium-arsenic combined pollution environments (e.g., Figure 2 (As shown).

[0029] The application of this tolerance ability is not limited to the experimental conditions described above. The strain exhibits the aforementioned tolerance characteristics in various soil or water environments with pH 5.0-8.5 and temperatures 25℃-37℃ when faced with cadmium and arsenic combined pollution, thus ensuring its practical application in complex environments.

[0030] Thirdly, this application also evaluated the removal capacity of the aforementioned Achromobacterium strain for cadmium and arsenic. This strain can generate secondary minerals such as iron (hydrogen) oxides through iron oxidation, effectively adsorbing or co-precipitating available cadmium and arsenic in the soil, thereby achieving simultaneous removal of these two heavy metals.

[0031] Taking the determination of iron oxidation capacity as an example, the specific experimental procedure is as follows: A liquid culture system was used in conjunction with tolerance and iron oxidation capacity tests. Different concentrations of cadmium and arsenic combined pollution treatments were set up in culture flasks containing basal culture medium (composition as described above) (Cd concentration gradients: 1 mg / L, 10 mg / L, 50 mg / L, and 100 mg / L; As(III) concentration gradients: 1 mg / L, 5 mg / L, 25 mg / L, and 50 mg / L). Freshly activated Achromobacterium seed culture was inoculated at a volume fraction of 5%, and a control group without inoculation but with the same contaminant concentration was established. All groups were cultured at 30℃ and 150 rpm in the dark with shaking for 96 h.

[0032] Samples were taken under aseptic conditions at 0h, 12h, 24h, 48h, 72h, and 96h of culture. After filtration through a 0.22μm filter to remove bacterial cells, the residual Fe in the filtrate was determined using the ferrozine colorimetric method. 2+ The concentration of total iron was determined by spectrophotometry or atomic absorption spectrometry using o-phenanthroline method, and the Fe concentration was calculated accordingly. 3+ Production amount and Fe 2+ Oxidation rate. The total residual concentrations of Cd and As in the solution were determined using inductively coupled plasma mass spectrometry (ICP-MS).

[0033] Compared with the blank control group, the Fe in the inoculated culture medium was higher. 2+ The total iron concentration decreased significantly with increasing culture time, accompanied by a change in the culture medium color from light green to reddish-brown (indicating the formation of Fe). 3+ Hydroxides (e.g.) Figure 3 As shown). Under simulated low-concentration combined pollution conditions (Cd≤50 mg / L, As≤50 mg / L), the concentrations of available Cd and As continuously decreased with incubation time. After 120 h of incubation, the removal rate of available As and Cd reached a maximum of 70% to 90% (e.g.). Figure 4 (As shown).

[0034] According to this application, iron oxidation capacity is the core mechanism by which this strain exerts its remediation function in the soil environment. When introduced into soil containing Fe... 2+ Minerals or soluble Fe 2+ When contaminated soil is exposed to pollutants, this ability can drive the transformation of iron forms and the formation of secondary minerals in the soil microenvironment, thereby providing the necessary physicochemical conditions for the in-situ fixation of cadmium and arsenic. This ability, combined with its aforementioned high tolerance, constitutes a unique technical advantage of this strain in the simultaneous remediation of cadmium and arsenic combined pollution.

[0035] Fourthly, this application provides a method for preparing a microbial agent containing the aforementioned *Achromobacterium* strain. The method, based on the physiological characteristics of the *Achromobacterium* strain, aims to efficiently obtain a highly active, high-density, and stable microbial agent product for the remediation of cadmium-arsenic contaminated soil.

[0036] The preparation method includes the following steps: Step 1: Strain activation and seed culture preparation The *Achromobacterium* strain preserved in glycerol tubes or slant agar at -80℃ was aseptically inoculated into liquid activation medium. The activation medium could be nutrient-rich LB medium (10 g / L trypsin, 5 g / L yeast extract, 5 g / L sodium chloride). The strain was incubated at 30℃-35℃ with static or shaking (100-150 rpm) for 24-48 hours to allow it to resume vigorous growth. An appropriate amount of the activated culture was then transferred to an Erlenmeyer flask containing 50-100 mL of liquid seed culture medium and incubated with shaking at 30℃-40℃ and 100-150 rpm for 12-24 hours, until the culture reached the late logarithmic growth phase (typically OD...). 600 A value between 1.0 and 2.0 indicates a highly active primary seed culture. To obtain a more uniform and viable inoculum, the primary seed culture can be transferred again to an activation medium at an inoculum volume of 1%-5% (v / v) for secondary expansion under the same culture conditions to obtain a seed culture for fermentation inoculation.

[0037] Step 2: Fermentation scale-up culture The *Achromobacterium* provided in this application can produce a large number of live *Achromobacterium* cells and / or fermentation products through fermentation culture. This application does not impose any particular limitation on the fermentation culture method, as long as the fermentation culture method can enable the *Achromobacterium* to proliferate in large quantities.

[0038] The seed culture prepared in step 1 is aseptically inoculated into a fermenter or large Erlenmeyer flask containing fermentation medium at an inoculation rate of 2%-10% (v / v). A preferred fermentation medium formulation comprises: a carbon source (e.g., glucose 2 g / L-5 g / L), a nitrogen source (e.g., tryptone 3 g / L-8 g / L, yeast extract 1 g / L-3 g / L, (NH4)2SO4 0.5 g / L-2 g / L), inorganic salts (KH2PO4 0.5 g / L-1.5 g / L, MgSO4·7H2O 0.2 g / L-0.5 g / L, CaCl2 0.05 g / L-0.2 g / L), and ferric ammonium citrate (5 g / L-20 g / L, preferably 10 g / L). The initial pH of the medium is adjusted to 6.5-7.5. Fermentation is carried out under controlled temperature, aeration, and stirring conditions. Specifically, the culture temperature is maintained at 30℃-37℃, and the stirring speed is set to 100-200 rpm depending on the container size to ensure sufficient dissolved oxygen. The culture time is generally 48-96 hours, and OD levels can be monitored. 600 The optimal harvest time can be determined by the value, pH, or residual sugar content.

[0039] Step 3: Collection of bacterial cells and preparation of inoculum After fermentation, the wet bacterial cells are collected by centrifugation or membrane filtration. The obtained bacterial cells are thoroughly mixed with a compound freeze-drying protectant under low-temperature aseptic conditions to prepare a bacterial suspension. The protectant contains nutritional protectants (such as trehalose and glycerol) and pH buffer salts, with the total amount added accounting for 5%-15% of the bacterial suspension mass. The uniformly mixed bacterial suspension is pre-frozen and then placed in a freeze dryer for freeze-drying under low-temperature vacuum conditions until the moisture content of the material is below 5%. The dried lumps are 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. 10 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.

[0040] Preferably, during the fermentation process in step 2, carbon sources (such as glucose) and iron salts can be added in batches via a feeding method to further increase the final cell density and iron oxidase activity.

[0041] As can be seen, the above preparation method is relatively simple in process and has controllable cost, and can stably produce efficient and highly active functional microbial agents, providing a reliable product guarantee for large-scale farmland soil remediation applications. This agent can be applied directly or used in combination with organic fertilizers, soil conditioners, etc.

[0042] Fifthly, this application utilizes the high tolerance, strong iron oxidation capacity, and cadmium-arsenic simultaneous passivation function driven by the strain to carry out bioremediation of soil contaminated by cadmium (Cd) and arsenic (As).

[0043] The specific implementation method is illustrated through the following examples: Take 1 kg of cadmium-arsenic contaminated farmland soil (pH 6.8, Cd content 5 mg / kg, As content 150 mg / kg) and place it in a test pot. Weigh 1.0 g of the freeze-dried powdered bacterial agent prepared in this application (viable count 3.5 × 10⁻⁶). 10 The bacterial suspension (CFU / g) was resuspended in sterile water and mixed thoroughly. 600 =1.2) Apply the bacterial suspension evenly to the soil. Specifically, spray the suspension evenly onto the soil surface, then mix thoroughly using sterile tools to ensure even contact between the bacterial agent and the 0-15 cm soil layer. A control group was set up, sprayed with an equal amount of sterile water; all other treatments were the same. The pots were placed in a controlled greenhouse for cultivation, maintaining a temperature of 25℃-30℃ during cultivation. Soil moisture was maintained at 60%-70% of field capacity by periodic weighing and water replenishment.

[0044] On the 60th day after treatment, soil samples were collected and mixed evenly. One part was immediately used to determine the concentration of Cd and As in the soil pore water (after centrifugation and filtration, the concentration was determined by ICP-MS); the other part was air-dried and ground, and the chemical speciation of Cd and As in the soil was systematically analyzed using the Tessier five-step continuous extraction method, with a focus on the changes in the content of available forms (i.e., exchangeable and carbonate-bound forms).

[0045] After 60 days, the proportion of available Cd (exchangeable and carbonate-bound) in the soil of the treated group decreased by 45% compared with the control group, and the proportion of available As decreased by 30%. The concentrations of Cd and As in the soil pore water decreased by 50% and 40%, respectively. The results indicate that the microbial agent of this application can effectively immobilize Cd and transform and fix As, significantly reducing its environmental risk.

[0046] Table 1 ; The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A bioremediation method for cadmium-arsenic co-contaminated soil, characterized in that, Includes the following steps: The target microbial agent was applied to the contaminated soil, and the biomineralization of Achromobacterium strain LMG 5911 was used to remove available cadmium and arsenic from the soil simultaneously, so as to achieve long-term immobilization of cadmium and arsenic. When the viable count of the bacterial agent is ≥1.0×10⁻⁶ 10 When the concentration of CFU / g is 0.0001 g / kg to 0.1 g / kg soil, the application rate of the microbial agent is 0.0001 g / kg to 0.1 g / kg soil.

2. The bioremediation method for cadmium-arsenic co-contaminated soil according to claim 1, characterized in that, The Achromobacter sp. LMG 5911 has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, on December 31, 2025, with accession number CCTCC NO: M 20253065. Its identification characteristic is Achromobacter sp. LMG-5911.

3. The bioremediation method for cadmium-arsenic co-contaminated soil according to claim 1, characterized in that, The target microbial inoculant is prepared through the following steps: The Achromobacterium strain LMG 5911 was activated and cultured to prepare a seed culture. The seed culture was inoculated into a fermentation medium containing ferrous salts for expanded fermentation culture to obtain a high-density bacterial suspension; The high-density bacterial suspension was mixed evenly with a freeze-drying protectant and then freeze-dried under vacuum to obtain the microbial agent.

4. The bioremediation method for cadmium-arsenic co-contaminated soil according to claim 3, characterized in that, The fermentation medium consists of the following components: tryptone 5.0 g / L-10.0 g / L, yeast extract 3.0 g / L-5.0 g / L, sodium chloride 5.0 g / L-10.0 g / L, and ferric ammonium citrate 0.5 g / L-5.0 g / L; the pH value is 6.5-7.

5.

5. The bioremediation method for cadmium-arsenic co-contaminated soil according to claim 3, characterized in that, The freeze-drying protectant comprises: trehalose at a w / v of 5%-10% and glycerol at a v / v of 1%-5%.

6. The bioremediation method for cadmium-arsenic co-contaminated soil according to claim 3, characterized in that, The step of activating and culturing the Achromobacterium strain LMG 5911 to prepare seed culture specifically includes: The Achromobacterium strain LMG 5911 was inoculated into liquid activation medium under aseptic conditions and cultured at 30℃-35℃ for 24 h-48 h to obtain activated culture. The activated culture was transferred to an Erlenmeyer flask containing 50 mL-100 mL of liquid seed culture medium and cultured with shaking at 30℃-40℃ and 100 rpm-150 rpm for 12 h-24 h, until the OD of the culture medium was reduced. 600 A value of 1.0-2.0 yields a primary seed solution; The primary seed culture was transferred again to activation medium at an inoculation rate of 1%-5% (v / v) for secondary expansion under the same culture conditions as the primary seed culture preparation, to obtain the seed culture; the OD of the seed culture... 600 The value is 1.5-3.

0.

7. The bioremediation method for cadmium-arsenic co-contaminated soil according to claim 6, characterized in that, The activation culture medium consists of the following components: 10 g / L trypsin, 5 g / L yeast extract, and 5 g / L sodium chloride.

8. The bioremediation method for cadmium-arsenic co-contaminated soil according to claim 3, characterized in that, The step of inoculating the seed culture into a ferrous salt-containing fermentation medium for expanded fermentation culture to obtain a high-density bacterial suspension includes: The seed culture is aseptically inoculated into a container containing fermentation medium at an inoculation rate of 2%-10% by volume and cultured at a temperature of 30℃-37℃, a stirring speed of 100 rpm-200 rpm, and a culture time of 48 h-96 h.

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

5.

10. The bioremediation method for cadmium-arsenic co-contaminated soil according to claim 3, characterized in that, The mixing volume ratio of the high-density bacterial suspension to the freeze-drying protectant is 1:1 to 2:1; The vacuum freeze-drying process conditions are as follows: the pre-freezing temperature is -40℃ to -80℃, and the pre-freezing time is 2 h to 6 h; the cold trap temperature for vacuum drying is -50℃ to -60℃, the vacuum degree is maintained at 10 Pa to 30 Pa, and the drying time is 24 h to 48 h, until the sample moisture content is less than 5%.

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