Alcaligenes 09 and application thereof

Abstract

The invention discloses an alcaligenes 09 and application thereof, the alcaligenes 09 is preserved in China Center for Type Culture Collection with the preservation number of CCTCC M 2025997, the 16S rDNA sequence of the alcaligenes 09 is shown as SEQ ID NO.1, and the alcaligenes 09 has a specific form and physiological and biochemical characteristics. The invention also discloses a liquid inoculant containing the strain and a preparation method of the liquid inoculant. The liquid inoculant is prepared through specific culture, fermentation and dilution steps. The degradation rate of the strain to 50 mg / L of albendazole within 72 h reaches 82.65%, the maximum tolerance concentration to cadmium is 80 mg / L, and the strain can be used for repairing water or soil co-polluted by albendazole and cadmium, has the advantages of high efficiency, environmental protection and low cost, and has important application value in environmental pollution treatment.

Description

Technical Field

[0001] The present invention relates to biological agriculture and related industries, in particular to the technical fields of microbial strains, enzyme engineering, etc., and particularly to a strain of Alcaligenes ( Alcaligenes sp.) 09, and its fermentation products and their application in degrading propanthazole residues and removing heavy metal cadmium. Background Art

[0002] Propylthiozole ( Albendazole ), chemical name is N-5-(propylthio)-1H-benzimidazol-2-yl) methyl carbamate, also known as albendazole, Shibaoling, propylthioamide and enterocin, etc. Because its structure has only one more propylthio group than carbendazim, it is also called propylthiocarbendazole. Its chemical formula is C 12 H 15 N3O2S, whose core structure is a benzimidazole ring, is connected to a propylthio group through a thioether bond, giving it a unique broad-spectrum biological activity - it has the dual functions of deworming humans and animals and preventing and controlling plant diseases.

[0003] When used preventively, propanthazole effectively protects plants from infection by inhibiting spore germination and pathogen invasion. It has a broad spectrum of activity, demonstrating effective control against diseases caused by Ascomycetes (such as powdery mildew and anthracnose), Basidiomycetes (such as rust and smut), and most Deuteromycetes (such as rice blast and anthracnose). It also has some effectiveness against downy mildew and late blight caused by flagellates.

[0004] Surprisingly, indoor bioassays and field trials have also revealed that propanthazole is also effective against bacterial and viral diseases. Furthermore, propanthazole promotes cell division and stimulates plant growth, enhancing plant vitality, accelerating plant greening, improving photosynthetic efficiency, and promoting calcium absorption in crops. In practical applications, crops treated with propanthazole often exhibit greener leaves and more beautiful fruits, higher quality, and healthier appearance, significantly increasing yields and incomes.

[0005] Studies have shown that propanthazole is biotoxic to non-target organisms and can cause adverse effects on the nervous, liver, immune, reproductive and hematopoietic systems after exposure ( Piloiu et al., American Journal of Therapeutics , 2021,28 (3): e335-e340).

[0006] Currently, propanthioazole is frequently detected in water environments around the world, including river water, sea water, domestic sewage and hospital sewage treatment plants ( Sim et al., Journal of Hazardous Materials , 2013, 248:219-227). Although usually at low concentrations (ng / L), propanthazole residues have been found even in residential drinking water, raising concerns about potential risks to non-target organisms ( , Science of the Total Environment, 2014, 468: 415-428). Due to its high octanol-water partition coefficient, high organic carbon partition coefficient and low water solubility, prothioconazole is prone to accumulate in soil and sediment (Wang et al., 2014, Environmental Pollution, 187: 66-73). Belew et al., Environmental Pollution , 2021, 269: 116106).

[0007] This persistence of prothioconazole is particularly pronounced in tropical and subtropical regions, where soil-borne helminth infections are more common and prothioconazole is heavily used, resulting in long-term environmental residues (Wang et al., 2014, Environmental Pollution, 187: 66-73). Schistosomiasis et al., Weekly Epidemiological Record , 2019, 95: 629-640). With the extensive use of prothioconazole in the farming and planting industry, the problem of its residues in soil and water cannot be ignored. Therefore, it is of great significance to seek new technologies for efficient, safe and economic management of pesticide residues and to solve the contradiction between pest control and pesticide residues.

[0008] On the other hand, cadmium is one of the most harmful heavy metals to animals, plants and human bodies in nature, and is collectively referred to as the "five poisons" in soil together with mercury, arsenic, chromium and lead. Cadmium is a non-essential element in human physiological processes. Environmental cadmium enters the human body through dietary intake, drinking contaminated water and other means, ultimately leading to kidney stones, diabetes and kidney failure and other problems, seriously endangering human health.

[0009] With the extensive application of cadmium in industrial and agricultural fields and the extension of human activity range, combined with the characteristics of cadmium that cannot be degraded by microorganisms, can migrate over long distances and is easily absorbed and accumulated by plants, cadmium pollution has seriously affected the sustainable development of China's agricultural production and human health. Therefore, the control of cadmium pollution has become a hot issue of concern, and it is urgent to control and remediate cadmium pollution.

[0010] In recent years, microbial remediation technology has become an effective measure for the treatment of various environmental pollutants due to its environmental protection, high efficiency, economy and environmental friendliness. This technology converts pesticide residues into harmless substances, passivates or adsorbs heavy metals, effectively reducing the pollution of pesticides and heavy metals to the environment and agricultural products, and helps to solve the ecological and environmental problems caused by pesticide and heavy metal contaminated soil or water. Currently, there is no report on a degradation strain that degrades prothioconazole and is resistant to cadmium.

[0011] Therefore, it is a very necessary work and a feasible way to selectively screen efficient degradation strains, study microbial remediation agents, and accelerate the removal of prothioconazole and cadmium in the environment by artificial inoculation to eliminate pollutants. SUMMARY

[0012] The application aims to provide a strain of Alcaligenes, a related bacterial agent, a preparation method and application, so as to efficiently degrade propyzamide residues and remove heavy metal cadmium, and solve the environmental pollution treatment problem caused by the two.

[0013] Based on the first main aspect of the application, a strain of Alcaligenes sp. 09 is provided, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC M 2025997, and the preservation date is May 8, 2025. Alcaligenes

[0014] The Alcaligenes sp. 09 is sent to Shanghai Shengong Bioengineering Co., Ltd. for 16S rDNA sequencing, and through BLAST retrieval, it is found that the homology of the 1439 bp sequence of the strain 09 and Alcaligenes sp. (ON920680) is 99%, and at the molecular level, it is determined as Alcaligenes sp. (ON920680). Alcaligenes sp. Alcaligenes sp The 16S rDNA sequence of the strain 09 is uploaded to the GenBank database, and the GenBank accession number is PV564662. At the same time, the strain is preserved in the China Center for Type Culture Collection (CCTCC), located in Wuhan, Wuhan University, China.

[0015] The 16S rDNA sequence of the strain is shown as SEQ ID NO. 1.

[0016] The morphological characteristics of the Alcaligenes sp. 09 are as follows: the strain is rod-shaped or short rod-shaped, with a size of 0.5-0.8 x 0.5-2.0 μm, the colony is smooth and viscous, opaque, grows fast, and the gram staining is negative; and the strain cannot ferment glucose, lactose, galactose, maltose, sucrose and cellobiose, but can ferment fructose, xylose, mannose, L-lactic acid, L-alanine, L-aspartic acid and L-malic acid; and the strain is negative in gelatin liquefaction, V-P, starch hydrolysis, indole and urease tests, and is positive in M-R, oxidase, contact enzyme and catalase tests, and cannot utilize nitrate.

[0017] Based on the second main aspect of the application, a liquid bacterial agent containing the aforementioned Alcaligenes sp. 09 or a fermentation product thereof is provided. Alcaligenes

[0018] As a further preferred scheme, the liquid bacterial agent takes the Alcaligenes sp. 09 as a living component, and is prepared by adding 2% glucose to the fermentation broth obtained by culturing and fermenting the Alcaligenes sp. 09, and then diluting to OD 600 0.6. Alcaligenes The liquid bacterial agent is prepared by adding 2% glucose to the fermentation broth obtained by culturing and fermenting the Alcaligenes sp. 09, and then diluting to OD 600 0.6.​​600 The UV spectrophotometer detected the concentration to be greater than 1.5.

[0019] According to the third main aspect of the present invention, a method for preparing the aforementioned liquid bacterial agent is provided, the method comprising the following steps: (1) Alcaligenes Alcaligenes sp.)09 Luria-Bertani The bacteria were cultured in LB medium at 30-35°C and 150-200 rpm with shaking until the logarithmic growth phase to obtain the strain. (2) Inoculate the bacterial strain into a seed bottle at an inoculum volume ratio of 1% of the culture medium volume, and culture with shaking at 30-35°C and 150-200 rpm until the logarithmic growth phase to obtain a seed solution; (3) inoculating the seed liquid into a fermentation medium at an inoculum volume ratio of 10%, and fermenting and culturing at 30-35°C and 150-200 rpm for 45-50 hours to obtain a fermentation liquid; (4) Add 2% glucose to the fermentation broth and dilute to OD 600 The concentration of 0.6 was used to obtain a liquid bacterial agent.

[0020] As a further preferred embodiment, in the aforementioned method for preparing the liquid bacterial agent, the culture temperature in steps (1), (2), and (3) is 35°C, the oscillation speed is 180 rpm, and the fermentation culture time in step (3) is 50 h.

[0021] According to the fourth main aspect of the present invention, a method for separating and screening Alcaligenes from soil is provided. Alcaligenes sp.) 09, comprising the following steps: (1) Taking soil samples that have been exposed to propanthazole and cadmium for a long time, culturing them in the initial culture medium to obtain culture solution; (2) transferring the culture solution to a selective medium containing propidazole and cadmium, gradually increasing the concentrations of propidazole and cadmium in the selective medium for enrichment culture to obtain an enriched bacterial solution; (3) spreading the enriched bacterial solution on a solid separation and purification medium containing propanthazole and cadmium, and picking a single colony for purification culture after cultivation; (4) The strain with the strongest ability to degrade propanthazole and remove cadmium among the purified strains was screened to obtain Alcaligenes ( Alcaligenes sp.09).

[0022] According to the fifth main aspect of the present invention, the aforementioned Alcaligenes ( Alcaligenes sp.)09 in the degradation of propanthazole residues and removal of heavy metal cadmium.

[0023] According to the sixth main aspect of the present invention, a bioremediation agent for propanthazole-cadmium co-contamination is provided, which comprises the aforementioned liquid bacterial agent and a necessary biocompatible carrier, wherein the liquid bacterial agent is Alcaligenes ( Alcaligenes sp.)09 is the active ingredient, OD 600 It is 0.6 and contains 2% glucose.

[0024] Alcaligenes of the present invention ( Alcaligenes sp.)09 has outstanding efficiency in degrading propanthazole, with a degradation rate of up to 82.65% for 50 mg / L propanthazole within 72 hours. It can also grow normally in a propanthazole concentration range of 50-800 mg / L and adapt to environments with different pollution levels. At the same time, its maximum tolerance to cadmium concentration is 80 mg / L, and it can function stably in cadmium-contaminated environments, achieving the synergistic removal of propanthazole and cadmium, thus overcoming the limitations of single pollutant control.

[0025] Secondly, the microbial preparation process of the present invention has practical value. The microbial preparation is prepared through standardized culture, fermentation and dilution steps. The culture conditions (30-35°C, 150-200 rpm) are mild and easy to control. The fermentation cycle is 45-50 hours. The OD 600 Key indicators such as the above can be detected by conventional instruments, which is convenient for large-scale production. The inoculum is diluted to OD after adding 2% glucose. 600 It can be used when the pH is 0.6. It has low preparation cost and easy operation, and is suitable for promotion and application in actual pollution control scenarios.

[0026] Third, the present invention has a wide range of applications and is environmentally friendly. Alcaligenes sp.) 09 and the liquid bacterial agent prepared from this strain can be directly used to repair water bodies, soil and other environmental media co-contaminated by propanthazole and cadmium, without the need to add highly toxic chemical reagents. Pollutants can be removed through the natural metabolism and adsorption of microorganisms to avoid secondary pollution, which is in line with the concept of green and environmental protection and effectively alleviates the damage to the ecosystem caused by traditional chemical treatment.

[0027] Finally, the Alcaligenes bacteria of the present invention ( Alcaligenes The isolation and screening method for 09 is scientific and reliable. By enriching the strain with gradually increasing concentrations of propanthazole and cadmium, combined with purification and screening, the resulting strain possesses strong degradation and pollution tolerance capabilities, providing a stable strain for subsequent applications. This method is highly reproducible and provides a reference paradigm for screening similar functional strains.

[0028] In summary, the present invention achieves efficient, environmentally friendly, and low-cost treatment of propanthazole and cadmium pollution through the collaborative innovation of strains, microbial agents, preparation, and application, and has important practical application value and theoretical significance in the fields of agricultural environmental restoration and ecological protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only show some embodiments of the present application, and for those skilled in the art, other drawings can be obtained according to these drawings without any creative effort.

[0030] Figure 1 The structure of propyzamide is shown; Figure 2 The chromatogram of the standard sample of propyzamide in one embodiment of the present application is shown; Figure 3 The phylogenetic tree of Alcaligenes sp. 09 in one embodiment of the present application is shown; Figure 4 The degradation ability of Alcaligenes sp. 09 to propyzamide and the tolerance to cadmium in one embodiment of the present application are shown; Figure 5 The optimization of the degradation conditions of Alcaligenes sp. 09 to propyzamide in one embodiment of the present application is shown; Figure 6 The removal ability of Alcaligenes sp. 09 to propyzamide and cadmium in one embodiment of the present application is shown. DETAILED DESCRIPTION

[0031] The preferred embodiments of the present application will be described in detail below with reference to the drawings, so as to more clearly understand the purpose, characteristics and advantages of the present application. It should be understood that the embodiments shown in the drawings are not a limitation on the scope of the present application, but only to illustrate the essential spirit of the technical solutions of the present application.

[0032] In the following description, for the purpose of explaining various disclosed embodiments, certain specific details are set forth in order to provide a thorough understanding of various disclosed embodiments. However, persons of ordinary skill in the relevant art will recognize that embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, structures, and techniques associated with the present application are not shown or described in order to avoid obscuring embodiments.

[0033] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. In addition, the particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0034] Example 1: Isolation and identification of Alcaligenes sp. 09.

[0035] (1) Isolation and screening of Alcaligenes sp. 09 Take 10 g of soil exposed to propyzamide and cadmium for a long time and add it to 50 mL of LB medium, cultivate at 30°C, 180 rpm / min for 24 h, take 10 mL of the above culture solution and add it to 100 mL of inorganic salt medium containing 50 mg / L of propyzamide and 10 mg / L of cadmium, after 72 h, transfer to a selection medium with higher concentrations of propyzamide and cadmium (propyzamide concentration increased by 50 mg / L each time, cadmium concentration increased by 10 mg / L each time), until the concentrations of propyzamide and cadmium reach 1000 mg / L and 200 mg / L respectively, and obtain the enriched degrading bacteria solution.

[0036] Take 50 μL of the above culture solution and spread it on a solid separation and purification medium with propyzamide and cadmium concentrations of 1000 mg / L and 200 mg / L respectively, cultivate at 28°C, wait for visible colonies to appear on the plate, pick single colonies and perform repeated streaking and purification.

[0037] Combine the same strains based on colony appearance and microscopic morphological characteristics, inoculate the purified strains into a slant solid separation and purification medium for preservation.

[0038] Determine the degradation ability of the purified strains to propyzamide and the removal ability of cadmium by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) and inductively coupled plasma mass spectrometry (ICP-MS), select the strain with the strongest removal ability (named 09) for subsequent research.

[0039] The calculation method of removal rate is as follows:

[0040] UPLC-MS / MS determination conditions: ultra-high performance liquid chromatography-triple quadrupole liquid chromatography-mass spectrometer (Agilent 1290II-G6470A) equipped with Eclipse Plus C 18 column (4.6 x 100 mm, 3.5 μm particle size); electrospray positive ion source: AJS ESI+, scanning mode is multiple reaction ion monitoring mode (MRM), sheath gas temperature 250°C, sheath gas flow rate: 11.0 L / min; carrier gas temperature 300°C, carrier gas flow rate: 5.1 L / min; capillary voltage: 3500 V, atomizer pressure: 15.0 psi.

[0041] The mass spectrometry parameters of propylthiouracil are shown in Table 1. The mobile phase is 0.1% formic acid aqueous solution (V / V) (A) - methanol (B), the flow rate is 0.3 mL / min, the injection volume is 5 μL, and the separation is performed by using binary gradient elution, and the gradient elution program is shown in Table 2. The retention time is 6.254 min, and the structural formula of propylthiouracil is shown in Figure 1 , and a typical chromatogram is shown in Figure 2 .

[0042] Table 1 Mass spectrometry parameters of propylthiouracil

[0043] " indicates the quantitative ion.

[0044] Table 2 Elution program of mobile phase of propylthiouracil in UPLC-MS / MS

[0045] ICP-MS determination conditions: ZB-5MS capillary column (30.0 mm x 0.25 mm x 0.25 μm), initial temperature at 50℃ for 2 min, then increased to 320℃ at a rate of 6℃ / min. Helium (≥99.999 as carrier gas, flow rate of 1.0 mL / min, injection volume of 1.0 μL. The ion source temperature and source voltage are set to 200 and 1124 V, respectively. The radio frequency power is set to 1200 W, and the flow rates of plasma gas, carrier gas, auxiliary gas and cell gas are 80, 0.70, 1.10 and 6.0 L / min, respectively.

[0046] (2) Identification of Alcaligenes 09 Morphological identification: The single colony state of strain 09 in the logarithmic growth phase and with stable colony size was described, mainly including the size, color, transparency and surface state of the colony. The results showed that strain 09 grew faster on LB solid medium, was rod-shaped or short rod-shaped, with a size of 0.5-0.8 x 0.5-2.0 μm, the colony was smooth and moist, not transparent, grew faster, slightly shiny, and the white colony was not sticky to silk and easy to pick.

[0047] Physiological and biochemical characteristics analysis: The physiological and biochemical characteristics of Alcaligenes 09 were determined by referring to "Microbiology Experiment" (Shen Ping, Fan Xiurong, Li Guangwu. Microbiology Experiment (3rd Edition). Beijing: Higher Education Press, 1999.), "Common Bacteria System Identification Manual" (Dong Xiuzhu, Cai Miaoying. Common Bacteria System Identification Manual. Beijing: Science Press, 2011.) and "Berger Bacteria Identification Manual" (R·E·Buckanan, N·E·Gibson. Berger's Identification Manual of Bacteriology. Beijing: Science Press, 1984).

[0048] ​The test results are shown in Table 3. Strain 09 showed negative Gram stain reaction and could not ferment glucose, lactose, galactose, maltose, sucrose and cellobiose, but could ferment fructose, xylose, mannose, L-lactic acid, L-alanine, L-aspartic acid and L-malic acid. The gelatin liquefaction, VP, starch hydrolysis, indole and urease tests were negative, and the MR, oxidase, catalase and catalase tests were positive, and the strain could not utilize nitrate.

[0049] Table 3 Physiological and biochemical characteristics of Alcaligenes 09

[0050] Note: "+" indicates a positive reaction, "-" indicates a negative reaction, VP indicates Voges-Proskauer, and MR indicates Methyl-Red.

[0051] Homology Analysis of the 16S rDNA of Strain 09: 16S rDNA gene was amplified and sequenced by PCR using universal bacterial primers 27F (5'-AGAGTTTGATC MTGGCTCAG-3') and 1492R (5'-GGTTACCTTG TTACGACTT-3'). The PCR product was sequenced by Shanghai Sangon Biotechnology Co., Ltd. The obtained 16S rDNA of strain 09 is shown in SEQ ID NO. 1.

[0052] The determined sequence was compared with the 16S rDNA sequence in the GenBank database using BLAST software at NCBI (accession number: PV564662), and it was found to be similar to Alcaligenes Alcaligenes sp. (ON920680) and other strains have a 99% similarity in 16SrDNA gene sequence.

[0053] From the phylogenetic tree ( Figure 3 ) It can be seen that strain 09 and Alcaligenes sp. (ON920680, KJ807184, PP320389, PV652521, PV652544, PV652577, AB681169, MN493881) were in the same branch. Combining morphological observation, physiological and biochemical characteristics and 16S rDNA gene sequence analysis, strain 09 was identified as Alcaligenes sp. Alcaligenes sp.).

[0054] The strain Alcaligenes 09 was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on May 8, 2025, with the accession number CCTCC M 2025997.

[0055] Example 2: Preparation of Alcaligenes 09 degradation agent: 1) Inoculate Alcaligenes 09 into an inorganic salt culture medium and culture at 35°C and 180 rpm with shaking until the logarithmic growth phase to obtain the strain; 2) Inoculate the strain into a seed bottle containing culture medium at a 1% inoculum volume (v / v) and incubate at 35°C and 180 rpm until the logarithmic growth phase to obtain seed solution. 3) The obtained seed liquid was inoculated into the fermentation medium at a volume ratio of 10%, and the fermentation culture was carried out at 35°C and 180 rpm for 50 h to obtain the fermentation liquid. The OD value of the fermentation liquid was measured by UV spectrophotometer. 600 If the OD value is greater than 1.5, the fermentation broth is directly diluted into liquid inoculum. 600 is 0.6.

[0056] Example 3: Determination of the degradation ability of Alcaligenes 09 to propanthazole and cadmium tolerance.

[0057] Add an appropriate amount of propanthazole acetone stock solution to a 50 mL conical flask. After the acetone is completely evaporated, add 1 mL of bacterial solution and 9 mL of inorganic salt (MSM) liquid culture medium to prepare a propanthazole concentration system of 50, 100, 200, 400, 800, 1200 and 1600 mg / L, respectively.

[0058] A control containing MSM liquid culture medium without bacterial suspension was also used. Three replicates were set up for each group. The samples were incubated in a constant-temperature shaking incubator at 35°C and 180 rpm. After 72 hours of incubation in the dark, the residual aprothiazole concentration in the samples was analyzed using UPLC-MS / MS.

[0059] In a 50 mL conical flask, 18 mL of LB medium and 2 mL of bacterial suspension were added, and a certain amount of 10 g / L CdCl2 solution was added to prepare a liquid system with cadmium concentrations of 20, 40, 60, 80, 100, 120, and 140 mg / L, respectively. Three replicates were set up for each group; 2 mL of sterile water was used instead of the bacterial suspension as a control. Then, the sample was placed in a constant temperature shaking box and samples were taken at regular intervals. The absorbance at 600 nm was measured using a UV-visible photometer, and the OD value was used. 600 express.

[0060] The results are as follows Figure 4 As shown in the figure, strain 09 can grow normally in the range of 50-800 mg / L of albendazole. When the albendazole concentration is greater than 1200 mg / L, the degradation rate of strain 09 is extremely low, which can be regarded as the inability of strain 09 to degrade albendazole or death.

[0061] Alcaligenes 09 grew normally when the cadmium concentration in the culture medium was between 20 and 80 mg / L. Subsequently, as the cadmium concentration increased, the strain's growth decreased significantly, indicating that cadmium inhibited the growth of strain 09. Furthermore, when the cadmium concentration exceeded 80 mg / L, strain 09's growth was significantly inhibited, indicating that its maximum tolerance to cadmium is 80 mg / L.

[0062] Example 4: Optimization of conditions for the degradation efficiency of propanthazole by Alcaligenes 09.

[0063] In order to optimize the degradation conditions of propanthazole by Alcaligenes 09, the initial pH was set at 5.0-9.0 (35°C), the culture temperature was 20-40°C (pH 7), and the bacterial concentration was 2-10×10 8 cfu / mL (pH 7, 35°C) and aprothiazole concentrations ranging from 25 to 125 mg / L (pH 7, 35°C). Alcaligenes 09 was cultured in 20 mL of inorganic salt medium containing 50 mg / L aprothiazole at 180 rpm / min. Each treatment was repeated three times. Inorganic salt medium without Alcaligenes 09 was used as a control. Aprothiazole was measured using UPLC-MS / MS at 12-h intervals, and the degradation rate was calculated based on the aprothiazole concentration in the control group. The formula for calculating the aprothiazole degradation rate is:

[0064] The results are as follows Figure 5 As shown, strain 09 had the highest degradation rate for albendazole at pH 7.0, reaching 81.45% after 72 hours of inoculation. At a culture temperature of 35°C, strain 09 had a degradation rate of 80.14% for albendazole. After 72 hours of inoculation, strain 09 had a degradation rate of 42.35% for 125 mg / L albendazole and 95.48% for 25 mg / L albendazole, indicating higher degradation rates at lower substrate levels. When the inoculation concentration of strain 09 was 6×10 8 cfu / mL, the degradation rate of propanthazole was the highest, reaching 82.37%. In summary, strain 09 had the highest degradation rate at pH 7.0, 30℃, and 6.0×10 8 cfu / mL, the degradation effect of propanthazole was the best.

[0065] Example 5: Determination of the removal ability of Alcaligenes 09 on propanthazole and cadmium.

[0066] Propylthiozole and cadmium were added to the inorganic salt culture medium to make the final concentrations of 50.0 mg / L and 10 mg / L, respectively. The bacteria were inoculated into the above-mentioned inorganic salt culture medium at an inoculum size of 10%, and the uninoculated culture medium was used as a control. The culture was cultured in the dark at 35°C (180 rpm) for 0-72 h, and samples were taken regularly.

[0067] The concentrations of prothioconazole and cadmium were determined by UPLC-MS / MS and ICP-MS, and the removal rates were calculated, and the results are shown in Table 1. Figure 6 As shown in Table 1, the Alcaligenes 09 can effectively remove prothioconazole and cadmium in a short time, and the removal rates of prothioconazole and cadmium reach 82.65% and 50.38% within 72 h, which has a good biological removal effect. This result shows that the Alcaligenes 09 provided by the present application can efficiently degrade prothioconazole and cadmium, and has a wide application prospect in repairing water and soil bodies contaminated by prothioconazole and cadmium.

[0068] The culture medium used in the above examples is as follows: Inorganic salt culture medium: KH2PO40.4 g, K2HPO40.4 g, NH4Cl 1 g, MgCl20.1 g, Na2SO41.425 g, FeSO4·7H2O 0.025 g, trace element solution 10 mL, distilled water to 1 L, pH 7.0.

[0069] The trace element solution is composed of 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, H3BO30.029 g, NaNO3·4H2O 0.074 g, and distilled water 1000 mL.

[0070] Separation and purification culture medium: prothioconazole is used as the only carbon source in the selective inorganic salt culture medium, and different concentrations are added according to the experimental design requirements, and the pH is 7.0 (20 g agar is added to the solid culture medium).

[0071] LB culture medium: tryptone 10.0 g, NaCl 10.0 g, yeast extract 5.0 g, distilled water 1000 mL, pH 7.0, and 20 g agar is added to the solid culture medium.

[0072] The above culture media are sterilized in a pressure cooker at 121℃ for 20-30 minutes.

[0073] In the above examples, the technical terms, technical principles or technical means related to the technical solutions of the present application are not described in detail, which are known to those skilled in the art or common means.

[0074] ​The above shows and describes the basic principles and main features of the present application and the advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above embodiments, and the above embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A strain of Alcaligenes ( Alcaligenes sp.) 09, characterized in that The strain was deposited in the China Center for Type Culture Collection with the deposit number CCTCC M 2025997 and the deposit date of May 8, 2025.

2. Alcaligenes according to claim 1 ( Alcaligenes sp.) 09, characterized in that The 16S rDNA sequence of the strain is shown in SEQ ID NO.

1.

3. Alcaligenes according to claim 1 ( Alcaligenes sp.) 09, characterized in that The strain is rod-shaped or short rod-shaped, 0.5-0.8 × 0.5-2.0 μm in size, with smooth, sticky, opaque colonies, rapid growth, and Gram-negative staining. Furthermore, the strain cannot ferment glucose, lactose, galactose, maltose, sucrose, and cellobiose, but can ferment fructose, xylose, mannose, L-lactic acid, L-alanine, L-aspartic acid, and L-malic acid; In addition, the strain was negative for gelatin liquefaction, VP, starch hydrolysis, indole and urease tests, but positive for MR, oxidase, catalase and catalase tests, and could not utilize nitrate.

4. A method comprising the alcaligenes bacillus according to claim 1 ( Alcaligenes sp.)09 or its fermentation product.

5. The liquid bacterial agent according to claim 4, characterized in that The liquid bacterial agent is based on Alcaligenes ( Alcaligenes sp.)09 is a living component, produced by Alcaligenes ( Alcaligenes The fermentation broth obtained by culture and fermentation was diluted to OD 600 The fermentation broth was prepared at an OD of 0.

6. 600 The UV spectrophotometer detected the concentration to be greater than 1.

5.

6. A method for preparing the liquid bacterial agent according to claim 4, characterized in that: The following steps are involved: (1) Alcaligenes Alcaligenes sp.)09 Luria-Bertani The culture medium was shaken at 30-35°C and 150-200 rpm until the logarithmic growth phase to obtain the strain; (2) Inoculate the bacterial strain into a seed bottle at an inoculum volume ratio of 1% of the culture medium volume, and culture with shaking at 30-35°C and 150-200 rpm until the logarithmic growth phase to obtain seed liquid; (3) inoculating the seed liquid into a fermentation medium at an inoculum volume ratio of 10%, and fermenting and culturing at 30-35°C and 150-200 rpm for 45-50 hours to obtain a fermentation liquid; (4) Add 2% glucose to the fermentation broth and dilute to OD 600 The concentration of 0.6 was used to obtain a liquid bacterial agent.

7. The method for preparing the liquid bacterial agent according to claim 6, wherein: The culture temperature in steps (1), (2) and (3) was 35°C and the shaking speed was 180 rpm; the fermentation culture time in step (3) was 50 h.

8. A method for isolating and screening Alcaligenes from soil Alcaligenes sp.) 09, characterized in that The following steps are involved: (1) Taking soil samples that have been exposed to propanthazole and cadmium for a long time, culturing them in the initial culture medium to obtain culture solution; (2) transferring the culture solution to a selective medium containing propidazole and cadmium, gradually increasing the concentrations of propidazole and cadmium in the selective medium for enrichment culture to obtain an enriched bacterial solution; (3) spreading the enriched bacterial solution on a solid separation and purification medium containing propanthazole and cadmium, and picking a single colony for purification culture after cultivation; (4) The strain with the strongest ability to degrade propanthazole and remove cadmium among the purified strains was screened to obtain Alcaligenes ( Alcaligenes sp.09).

9. The Alcaligenes bacillus according to claim 1 ( Alcaligenes sp.)09 in the degradation of propanthazole residues and removal of heavy metal cadmium.

10. A bioremediation agent for propanthazole-cadmium co-contamination, characterized in that: The invention comprises the liquid bacterial agent according to claim 4, and a necessary biocompatible carrier, wherein the liquid bacterial agent is Alcaligenes ( Alcaligenes sp.)09 is the active ingredient, OD 600 It is 0.6 and contains 2% glucose.

Images