Alcaligenes faecalis X4 and application thereof

Abstract

The application discloses an Alcaligenes faecalis strain X4 and application thereof, the strain has been preserved in the China Center for Type Culture Collection (CCTCC) on May 8, 2025, is located in Wuhan, Wuhan University, China, and the preservation number is CCTCC M 2025998. The Alcaligenes faecalis strain X4 provided in the application has a degradation rate of more than 70% to 50 mg / L flumorph within 72 h, and the maximum tolerance concentration of cadmium is 120 mg / L. The strain provides strong technical support for the treatment of flumorph and cadmium compound pollution, and has important significance for solving complex environmental pollution problems.

Description

Technical Field

[0001] This invention relates to bio-agriculture and related industries, particularly to the fields of microbial strains and fermentation engineering, and especially to a strain of Alcaligenes faecalis (…). Alcaligenes faecalis X4 and its fermentation products and applications. Background Technology

[0002] Fluoromorphine ( Flumorph Fluoromorpholine, also known as floccomorph, has the chemical name 4-[3-(3,4-dimethoxyphenyl)-3-(4-fluorophenyl)acryloyl]morpholine. Fluoromorpholine is a colorless crystalline solid with a characteristic odor. It is an alkaline substance, readily soluble in acetone and ethyl acetate, and stable to light and heat under normal conditions. Fluoromorpholine is based on dimethomorpholine, with fluorine atoms replacing the chlorine atoms in dimethomorpholine. Its biological activity is significantly higher than that of dimethomorpholine, primarily inhibiting spore germination and thus suppressing pathogenicity. It exhibits excellent biological activity against diseases caused by oomycete pathogens, such as cucumber downy mildew, grape downy mildew, tobacco blight, and pepper blight.

[0003] Although flumorpholine is highly effective in controlling a variety of crop diseases, mounting evidence confirms that it is a toxic pollutant in the agricultural-soil-aquatic ecosystem, posing a risk to non-target organisms. Furthermore, flumorpholine has a relatively long half-life in ecosystems, ranging from 99 to 158 days in soil, and is very stable in buffered aqueous solutions and natural water, failing to degrade under dark conditions. The extensive use of flumorpholine in agriculture leads to exposure to flumorpholine residues, posing a significant threat to organisms, humans, and ecosystems. Therefore, there is an urgent need to develop eco-friendly and low-cost strategies for degrading flumorpholine pollutants.

[0004] On the other hand, due to unreasonable production and discharge practices such as industrial wastewater discharge, mining and smelting, agricultural activities, and urban sewage, heavy metals (most of which are teratogenic, carcinogenic, mutagenic, and non-degradable) have polluted rivers and groundwater. Cadmium ( Cadmium Cadmium (Cd) is a pervasive heavy metal in the natural environment that is toxic to humans, animals, and plants. It is found in almost all soil, surface water, and plant matter, and is considered one of the most dangerous substances. Cadmium pollution disrupts the balance of environmental ecosystems and impacts biodiversity. Cadmium compounds are more soluble than many other heavy metals, making them more readily absorbed by plants and accumulating in different edible parts of plants. Through the food chain, it can accumulate and enter the human body, leading to serious health problems such as kidney damage, osteoporosis, neurological problems, emphysema, osteomalacia, and even cancer with long-term exposure.

[0005] Cadmium pollution exists to varying degrees in countries worldwide, with soils in areas with sedimentary rocks, particularly carbonate rocks, exhibiting geochemically anomalous high background cadmium levels. In recent years, with the widespread use of cadmium in industry and agriculture and the expansion of human activities, coupled with cadmium's characteristics of being non-degradable by microorganisms, capable of long-distance migration, and easily absorbed and accumulated by plants, cadmium pollution has impacted the sustainable development of agricultural production and human health. Therefore, the control and remediation of cadmium pollution has become a major concern.

[0006] Currently, methods for removing pollutants from the environment (water and soil) mainly include physical, chemical, and biological methods. Biological methods primarily utilize microbial absorption. Microbial remediation technology, with its advantages of being environmentally friendly, efficient, economical, and environmentally friendly, has been widely applied in real life. This technology uses the adsorption and metabolic capabilities of microorganisms to convert pesticide residues into harmless substances and passivate or adsorb heavy metals in the soil so they are not absorbed by plants, effectively reducing pesticide and heavy metal pollution of the environment and agricultural products. It helps solve the ecological and environmental problems caused by pesticide and heavy metal pollution of soil or water bodies.

[0007] However, there are currently no microbial agents or products specifically designed to degrade flumorph and chlorine. Therefore, targeted screening of highly efficient degrading strains, research on microbial remediation agents, and the use of artificial inoculation to accelerate the degradation of bactericides and the passivation or adsorption of heavy metals in the environment to eliminate pollutants are essential and feasible approaches. Summary of the Invention

[0008] The purpose of this invention is to provide a strain of Alcaligenes faecalis ( Alcaligenes faecalis X4 and its liquid bacterial agent and applications are used to simultaneously degrade flumorpholine pollutants in the environment and passivate or adsorb cadmium pollutants, in order to solve the ecological restoration problem of water bodies and soil co-polluted by flumorpholine and cadmium.

[0009] Based on the first main aspect of the present invention, a strain of Alcaligenes faecalis ( Alcaligenes faecalis X4, characterized in that the strain is deposited at the China Center for Type Culture Collection, with accession number CCTCC M 2025998.

[0010] Furthermore, the 16S rDNA sequence of this strain is shown in SEQ ID NO.1.

[0011] Furthermore, the morphological and physiological-biochemical characteristics of this strain satisfy the following:

[0012] It is rod-shaped or short rod-shaped, with a size of 0.6~1.0×0.5~2.5 μm;

[0013] It shows a negative Gram staining reaction and cannot ferment sugars;

[0014] The catalase and oxidase tests were positive, while the indole, MR, VP, amylase, and gelatin liquefaction tests were negative.

[0015] It can utilize nitrates, but not nitrites.

[0016] The sugars mentioned include at least glucose, lactose, maltose, mannitol, and sucrose.

[0017] Based on a second key aspect of the present invention, a liquid bacterial agent is provided, comprising the aforementioned *Alcaligenes faecalis* (…). Alcaligenes faecalis X4 or its fermentation product as the living component, the OD of the liquid bacterial agent 600 It is 0.6.

[0018] Based on a third key aspect of the present invention, a method for preparing the aforementioned liquid bacterial agent is provided, comprising the following steps:

[0019] (1) Inoculate Alcaligenes faecalis X4 into beef extract peptone medium and culture at 30-35℃ and 150-200 rpm until the logarithmic growth phase to obtain the strain;

[0020] (2) Inoculate the bacterial strain obtained in step (1) into the seed bottle at an inoculation rate of 1% of the culture medium volume, and culture it at 30~35℃ and 150~200 rpm until the logarithmic growth phase to obtain the seed liquid;

[0021] (3) Inoculate the seed culture obtained in step (2) into the fermentation medium at an inoculation rate of 10% by volume, and ferment at 30-35℃ and 150-200 rpm for 45-50 h to obtain OD. 600 For fermentation broth with an OD value >1.5, add 2% glucose and dilute to an OD value of [missing value]. 600 The concentration was 0.6, resulting in a liquid bacterial agent.

[0022] As a further preferred option, in the aforementioned preparation method, the culture time of the logarithmic growth phase in steps (1) and (2) is 12~24 h.

[0023] Based on the fourth main aspect of the present invention, the aforementioned Alcaligenes feces ( Alcaligenes faecalis The application of X4 in the treatment of co-contamination of flumorpholine and cadmium includes inoculating the strain into environmental media contaminated with flumorpholine and cadmium for the degradation of flumorpholine and the passivation or adsorption of cadmium.

[0024] As a further preferred embodiment, the aforementioned applications also include applying the strain in liquid form to contaminated soil or water bodies.

[0025] Compared to existing technologies, which lack microorganisms capable of simultaneously degrading flumorpholine and tolerating cadmium, the present invention's strain X4 achieves a breakthrough in "dual-effect" bioremediation, simultaneously achieving highly efficient degradation of flumorpholine and high tolerance and removal of cadmium. Specifically, regarding highly efficient flumorpholine degradation, strain X4 achieves a degradation rate of 72.65% for 50 mg / L flumorpholine within 72 hours, far exceeding its half-life of 99-158 days in the natural environment. Furthermore, regarding high tolerance and removal of cadmium, strain X4 tolerates a maximum cadmium concentration of 120 mg / L, achieving a cadmium removal rate of 54.93% in co-polluted systems. This synergistic effect fills a technological gap in the bioremediation of complex pollution.

[0026] Meanwhile, the solution of this invention can significantly improve remediation efficiency and reduce costs. Compared with physical / chemical methods (such as adsorbent replacement and chemical precipitation), this invention has several unique advantages. First, the liquid bacterial agent prepared by this invention is easy to use; the liquid bacterial agent (OD) can be directly added. 600 =0.6) to the contaminated medium, no complicated equipment is required; at the same time, it can take effect quickly, significantly reducing pollutants within 72 hours. In addition, the microbial agent of the present invention is produced by fermentation using inexpensive culture medium (such as beef extract peptone, inorganic salts), and the cost is less than 20% of that of chemical treatment, which has good economic benefits.

[0027] The solution of this invention also achieves dual protection of environmental compatibility and safety. In this invention, the strain converts flumorpholine into a harmless substance through metabolism and fixes cadmium through passivation / adsorption, avoiding the secondary risks of chemical remediation and achieving zero secondary pollution. Moreover, the strain of this invention can work efficiently in a neutral environment (pH 7.0–8.0) and at room temperature (30℃), making it suitable for environmental media such as farmland soil and water bodies, and exhibiting good ecological adaptability; furthermore, the strain of this invention originates from the natural environment and carries no risk of genetic modification.

[0028] In summary, the solution presented in this invention is of great significance in promoting sustainable agricultural development. In application, it can reduce the absorption of cadmium by crops, ensuring food security at the source and blocking food chain contamination. Soil remediated using this invention is safe for cultivation, and the live bacteria in the microbial agent can continuously reproduce and exert degradation effects. Therefore, this invention solves the problems of two types of pollutants with a single microbial strain, combining high efficiency, economy, and ecological safety, providing an irreplaceable technical solution for the treatment of co-polluting fluoromorph and cadmium. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0030] Figure 1 The structural formula of fluoromorpholine is shown;

[0031] Figure 2 A typical chromatogram of flumorpholine in one embodiment of the present invention is shown;

[0032] Figure 3 A phylogenetic tree of Alcaligenes faecalis X4 is shown in one embodiment of the present invention;

[0033] Figure 4 A schematic diagram illustrating the degradation ability of Alcaligenes faecalis X4 to flumorpholine and its tolerance to cadmium in one embodiment of the present invention is shown.

[0034] Figure 5 This diagram illustrates the optimization of optimal degradation conditions for flumorpholine by Alcaligenes faecalis X4 in one embodiment of the present invention.

[0035] Figure 6 A schematic diagram illustrating the removal rates of flumorpholine and cadmium by *Alcaligenes faecalis* X4 in one embodiment of the present invention is shown. Detailed Implementation

[0036] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, so as to better understand the purpose, features and advantages of the present invention. It should be understood that the embodiments shown in the drawings are not intended to limit the scope of the present invention, but are only for illustrating the essential spirit of the technical solution of the present invention.

[0037] In the following description, certain specific details are set forth for the purpose of illustrating various disclosed embodiments in order to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the art will recognize that embodiments may be practiced without one or more of these specific details. In other instances, well-known apparatuses, structures, and techniques associated with this application may not have been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0038] Throughout this specification, references to "an embodiment" or "an embodiment" indicate that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Therefore, the appearance of "in an embodiment" or "an embodiment" in various places throughout the specification does not necessarily refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in any manner in one or more embodiments.

[0039] Example 1: Isolation and identification of Alcaligenes faecalis X4.

[0040] (1) Isolation and screening of Alcaligenes faecalis X4

[0041] 10 g of soil that had been exposed to flumorpholine and cadmium for a long period of time was weighed and added to 50 mL of beef extract peptone medium. The medium was incubated at 30 °C and 180 rpm / min for 24 h. 10 mL of the above culture solution was added to 100 mL of inorganic salt medium (containing 50 mg / L flumorpholine and 10 mg / L cadmium) and incubated. After 5 days, the medium was transferred to a selective medium with higher concentrations of flumorpholine and cadmium (the concentration of flumorpholine was increased by 50 mg / L each time, and the concentration of cadmium was increased by 10 mg / L each time) until the concentrations of flumorpholine and cadmium reached 1000 mg / L and 200 mg / L, respectively, to obtain an enriched degrading bacterial solution.

[0042] Spread 50 µL of the above culture solution onto solid separation and purification medium containing 1000 mg / L flumorpholine and 200 mg / L cadmium, respectively. Incubate at 28°C. Once visible colonies appear on the plates, pick single colonies and perform repeated streaking purification. Combine strains with the same colony morphology based on both appearance and microscopic characteristics. Inoculate the purified strains into slant solid separation and purification medium for later use.

[0043] The removal capabilities of the purified strains for flumorpholine and cadmium were determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) and inductively coupled plasma mass spectrometry (ICP-MS). The strain with the strongest removal capability (named X4) was selected for further research.

[0044] UPLC-MS / MS determination conditions: Ultra-high performance liquid chromatography-triple quadrupole LC-MS system (Agilent 1290II-G6470A), equipped with Eclipse Plus C 18Column (4.6 × 100 mm, 3.5 µm particle size); electrospray positive ionization source: AJS ESI+, scanning mode: multiple reaction monitoring (MRM); sheath gas temperature: 250 ºC, sheath gas flow rate: 11.0 L / min; carrier gas temperature: 300 ºC, carrier gas flow rate: 5.1 L / min; capillary voltage: 3500 V, nebulizer pressure: 15.0 psi. Quantitative ion pair: 372.2 / 285; qualitative ion pair: 372.2 / 165; collision energy: 20 eV / 40 eV; declustering voltage: 130 V. The mobile phase was 0.1% formic acid aqueous solution (V / V) (A)-methanol (B), and a gradient elution program was used for analysis: 0-3 min (30% A), 3-5 min (10% A), 5-8 min (90% A), 8-11 min (70% A), 11-15 min (30% A), at a flow rate of 0.5 mL / min and a column temperature of 30 °C; the injection volume was 10 μL. Retention times: 7.165 min (Z), 8.322 min (E). The structural formula of flumorpholine is shown below. Figure 1 Typical chromatograms are shown below. Figure 2 .

[0045] ICP-MS measurement conditions: On a ZB-5MS capillary column (30.0 mm × 0.25 mm × 0.25 μm), the initial temperature was maintained at 50 °C for 2 min, then increased to 320 °C at a rate of 6 °C / min. Helium (≥99.999) was used as the carrier gas at a flow rate of 1.0 mL / min, with an injection volume of 1.0 μL. The ion source temperature and source voltage were set to 200 °C and 1124 V, respectively. The RF power was set to 1200 W, and the flow rates of plasma gas, carrier gas, auxiliary gas, and cell gas were 80, 0.70, 1.10, and 6.0 L / min, respectively.

[0046] (2) Identification of Alcaligenes faecalis X4

[0047] Morphological identification: The purified strain X4, which is in the logarithmic growth phase and has a stable colony size, was described as a single colony, mainly including the size, color, transparency, and surface condition of the colony.

[0048] The results showed that strain X4 grew rapidly on beef extract peptone solid medium, forming rod-shaped or short rod-shaped colonies with a size of 0.6~1.0×0.5~2.5 μm. The colonies were smooth, moist, opaque, and grew quickly.

[0049] Physiological and biochemical characteristics analysis: The physiological and biochemical characteristics of Alcaligenes faecalis X4 were determined with reference to "Experimental Microbiology", "Manual of Systematic Identification of Common Bacteria" and "Bergey's Manual of Bacterial Identification".

[0050] The test results are shown in Table 1. The Gram staining reaction of strain X4 was negative, and it could not ferment sugars (glucose, lactose, maltose, mannitol and sucrose). The catalase and oxidase tests were positive, while the indole, MR, VP, amylase and gelatin liquefaction tests were negative. It could utilize nitrates but could not utilize nitrites.

[0051] Table 1. Physiological and biochemical characteristics of strain X4

[0052]

[0053] Note: "+" indicates a positive reaction, and "-" indicates a negative reaction.

[0054] Homology analysis of 16S rDNA of strain X4: 16S rDNA gene PCR amplification and sequencing were performed using universal bacterial upstream and downstream primers 27F (5'-AGAGTTTGATC MTGGCTCAG-3') and 1492R (5'-GGTTACCTTG TTACGACTT-3'). The PCR products were sequenced by Shanghai Sangon Biotech Co., Ltd.

[0055] The 16S rDNA of strain X4 was obtained as shown in SEQ ID NO.1. The determined sequence was compared for homology with 16S rDNA sequences in the GenBank database (accession number PQ867866) using BLAST software on NCBI, revealing a similarity to *Alcaligenes faecalis*. Alcaligenes faecalis The 16S rDNA gene sequence similarity of strains such as (JX276778) reached 99%.

[0056] From the phylogenetic tree ( Figure 3 It can be seen that strain X4 and Alcaligenes faecalis (JX276778) belongs to the same branch. Based on morphological observation, physiological and biochemical characteristics, and 16S rDNA gene sequence analysis, strain X4 was identified as Alcaligenes faecalis (JX276778). Alcaligenes faecalis This strain is *Alcaligenes faecalis* (…). Alcaligenes faecalis X4 was deposited on May 8, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, accession number CCTCCM2025998.

[0057] Example 2: Preparation of Alcaligenes faecalis X4 degradation agent:

[0058] 1) Inoculate 4 alkalobacterium feces with inorganic salt medium and culture at 30°C and 180 rpm with shaking until the logarithmic growth phase to obtain the bacterial strain;

[0059] 2) Inoculate the bacterial strain at a rate of 1% (V / V) into a seed bottle containing culture medium and incubate at 30℃ and 180 rpm until the logarithmic growth phase to obtain the seed culture;

[0060] 3) The obtained seed culture was inoculated into the fermentation medium at a volume ratio of 10%, and fermented at 30℃ and 180 rpm for 50 h to obtain the fermentation broth. The OD of the fermentation broth was measured by ultraviolet spectrophotometer. 600 If the OD value is greater than 1.5, the fermentation broth is directly diluted into a liquid inoculum. The OD value of the liquid inoculum is... 600 It is 0.6.

[0061] Example 3: Determination of the degradation ability of Alcaligenes faecalis X4 on flumorpholine and its tolerance to cadmium.

[0062] Add an appropriate amount of flumorpholine acetone stock solution to a 50 mL Erlenmeyer flask. After the acetone has completely evaporated, add 1 mL of bacterial culture and 9 mL of inorganic salt (MSM) liquid culture medium to prepare flumorpholine concentrations of 50, 100, 200, 400, 800, 1200 and 1600 mg / L.

[0063] Meanwhile, MSM liquid medium without bacterial suspension was set as a control. Each group was divided into three replicates. The samples were placed in a constant temperature shaking incubator and cultured at 30℃ and 180 rpm in the dark for 72 h. The concentration of residual flumorpholine in the samples was then analyzed by UPLC-MS / MS.

[0064] In addition, 18 mL of beef extract peptone liquid culture medium and 2 mL of bacterial suspension were added to a 50 mL Erlenmeyer flask, and a certain amount of 10 g / L CdCl2 solution was added to prepare liquid systems with cadmium concentrations of 30, 60, 90, 120 and 150 mg / L, with 3 replicates for each group; 2 mL of sterile water was used as a control instead of bacterial suspension.

[0065] Then, the sample was placed in a constant-temperature shaking oven, and samples were taken periodically. The absorbance at 600 nm was measured using a UV-Vis spectrophotometer, and the result was analyzed using OD. 600 express.

[0066] The results are as follows Figure 4As shown, strain X4 can grow normally within a flumorpholine concentration range of 50–800 mg / L, exhibiting a high degradation rate of flumorpholine. However, when the flumorpholine concentration exceeds 1200 mg / L, the degradation rate of flumorpholine by strain X4 is extremely low, indicating that strain X4 has no ability to degrade flumorpholine or is dead at this level. When the cadmium concentration in the culture medium is between 30–120 mg / L, *Alcaligenes faecalis* X4 can grow normally. Subsequently, as the cadmium concentration increases, the growth of the strain decreases significantly, indicating that cadmium inhibits the growth of strain X4. Furthermore, when the cadmium concentration exceeds 150 mg / L, the growth of strain X4 is significantly inhibited, indicating that the maximum tolerated cadmium concentration is 120 mg / L.

[0067] Example 4: Optimization of degradation conditions of flumorpholine by Alcaligenes faecalis X4.

[0068] To optimize the degradation conditions of flumorpholine by strain X4, the initial pH was set at 5.0–9.0 (30℃), the culture temperature at 25–45℃ (pH 7), and the concentration of strain X4 at 2–10 × 10⁻⁶. 8 CFU / mL (pH 7, 30℃), flumorpholine concentration 25–125 mg / L (pH 7, 30℃), strain X4 was cultured in 20 mL of inorganic salt medium containing 50 mg / L flumorpholine at 180 rpm / min, with each treatment repeated four times. Inorganic salt medium without strain X4 served as a control. Flumorpholine content was determined by UPLC-MS / MS at 12-hour intervals, and the degradation rate was calculated based on the flumorpholine content in the control. The calculation formula is as follows:

[0069] Degradation rate (%) = ×100%

[0070] The results are as follows Figure 5 As shown, strain X4 exhibited the highest degradation rate of flumorph at pH 7.0, reaching 70.88% after 72 h of inoculation, followed by pH 8.0 with a degradation rate of 67.48%. The degradation rate of flumorph decreased when the pH was below 7.0 or above 8.0, indicating that strain X4 was more effective at degrading flumorph under neutral to weakly alkaline conditions (pH 7.0–8.0). Strain X4 achieved the highest degradation rate of flumorph at 30℃, reaching 68.75%. After 72 h of inoculation, the degradation rate of 125 mg / L and 25 mg / L flumorph by strain X4 differed by 30.42%, indicating higher degradation efficiency at lower substrate levels. When the inoculation concentration of strain X4 was 6 × 10⁻⁶, the degradation rate was significantly higher. 8 At a concentration of cfu / mL, the degradation rate of flumorpholine was 69.17%. In conclusion, strain X4, at pH 7.0, temperature 30℃, and cell concentration of 6.0 × 10⁻⁶, showed optimal degradation. 8 The degradation ability of flumorpholine is optimal at a concentration of cfu / mL.

[0071] Example 5: Removal capacity of Alcaligenes faecalis X4 for flumorpholine and cadmium pollution

[0072] Fluoromorph and cadmium were added to an inorganic salt culture medium to achieve final concentrations of 50 mg / L and 10 mg / L, respectively. Bacterial cells were inoculated into the aforementioned inorganic salt culture medium at a 10% inoculum rate, with an uninoculated medium serving as a control. All samples were incubated at 30°C, pH 7.0, and a cell concentration of 6 × 10⁻⁶ cells / mL. 8 Incubate in the dark for 0–72 h at cfu / mL and 180 rpm, taking samples periodically.

[0073] The contents of flumorpholine and cadmium were determined using UPLC-MS / MS and ICP-MS methods, and the removal rates were calculated. Figure 6 As shown, Alcaligenes faecalis X4 achieved removal rates of 70.26% and 54.93% for flumorpholine and cadmium, respectively, within 72 h, indicating that this strain has highly efficient removal performance.

[0074] This result demonstrates that strain X4 provided by this invention can efficiently remove flumorpholine and cadmium, and has broad application potential in remediating soil and water bodies contaminated with flumorpholine and cadmium.

[0075] The culture medium used in the above embodiments is as follows:

[0076] Inorganic salt culture medium: KH₂PO₄ 0.4 g, K₂HPO₄ 0.4 g, NH₄Cl 1 g, MgCl₂ 0.1 g, Na₂SO₄ 1.425 g, FeSO₄·7H₂O 0.025 g, trace element solution 10 mL, diluted to 1 L with distilled water, pH 7.0. The trace element solution composition is as follows: ZnSO₄·7H₂O 1.1 g, MgSO₄·H₂O 0.58 g, (NH₄)₆Mo₇O₇ 24 ·4H2O 0.18 g, CoSO4·7H2O 0.024 g, CuSO4·5H2O 0.077 g, H3BO3 0.029 g, NaNO3·4H2O 0.074 g, distilled water 1000 mL.

[0077] Isolation and purification medium: Fluoromorph is used as the sole carbon source in selective inorganic salt medium. Different concentrations are added according to the experimental design requirements, pH 7.0 (20 g agar is added to solid medium).

[0078] Beef extract peptone medium: 3.0 g beef extract, 5.0 g NaCl, 10.0 g peptone, 1000 mL distilled water, pH 7.0, and 20 g agar added to the solid medium.

[0079] All the above culture media were sterilized in an autoclave at 121°C for 20–30 minutes.

[0080] The technical terms, principles, or means related to the technical solutions of the present invention mentioned in the above embodiments, which are not described in detail above, are all well-known technologies or common practices that are known to those skilled in the art.

[0081] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A strain of Alcaligenes faecalis ( Alcaligenes faecalis X4, characterized in that, This strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2025998; the 16S rDNA sequence of this strain is shown in SEQ ID NO.

1.

2. A liquid bacterial agent, characterized in that, Contains the Alcaligenes faecalis as described in claim 1 ( Alcaligenes faecalis X4 is the living component, and the OD of the liquid bacterial agent is... 600 It is 0.

6.

3. A method for preparing the liquid bacterial agent according to claim 2, characterized in that, Includes the following steps: (1) Inoculate Alcaligenes faecalis X4 into beef extract peptone medium and culture at 30-35℃ and 150-200 rpm until the logarithmic growth phase to obtain the strain; (2) Inoculate the bacterial strain obtained in step (1) into the seed bottle at an inoculation rate of 1% of the culture medium volume, and culture it at 30~35℃ and 150~200rpm with shaking until the logarithmic growth phase to obtain the seed liquid; (3) Inoculate the seed culture obtained in step (2) into the fermentation medium at an inoculation rate of 10% by volume, and ferment at 30-35℃ and 150-200rpm for 45-50 h to obtain OD. 600 For fermentation broth with an OD value >1.5, add 2% glucose and dilute to an OD value of [missing value]. 600 The concentration was 0.6, resulting in a liquid bacterial agent.

4. The method for preparing the liquid bacterial agent according to claim 2, characterized in that, The culture time for the logarithmic growth phase described in steps (1) and (2) is 12-24 h.

5. The method for preparing the liquid bacterial agent according to claim 3, characterized in that, The fermentation medium is an inorganic salt medium with the following composition: KH2PO4, 0.4 g; K2HPO4, 0.4 g; NH4Cl, 1 g; MgCl2, 0.1 g; Na2SO4, 1.425 g; FeSO4·7H2O, 0.025 g; 10 mL of trace element solution; and distilled water to a final volume of 1 L, pH 7.

0. The trace element solution composition is as follows: ZnSO4·7H2O, 1.1 g; MgSO4·H2O, 0.58 g; (NH4)6Mo7O 24 ·4H2O, 0.18 g; CoSO4·7H2O, 0.024 g; CuSO4·5H2O, 0.077 g; H3BO3, 0.029 g; NaNO3·4H2O, 0.074 g; 1000 mL distilled water.

6. The *Alcaligenes faecalis* as described in claim 1 (… Alcaligenes faecalis The application of X4 in the treatment of co-polluting flumethrin and cadmium is characterized by, The strain was inoculated into environmental media contaminated with flumorpholine and cadmium for the degradation of flumorpholine and the passivation or adsorption of cadmium.

7. The *Alcaligenes faecalis* according to claim 6 ( Alcaligenes faecalis The application of X4 in the treatment of co-polluting flumethrin and cadmium is characterized by, It also includes applying the strain in liquid form to contaminated soil or water.

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