Curtobacterium sp. and application thereof in degrading sulfachloropyridazine

Abstract

This invention discloses a strain of *Tridentium flexneri* and its application in the degradation of sulfachlorpyridazine. *Tridentium flexneri* (… Ancylobacter The preservation information for *Sp.* SAs-18 is as follows: Depository Institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC); Deposit Date: November 25, 2025; Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, Postcode: 510070; Accession Number: GDMCC No. 67354. This invention isolates a strain of *Vibrio parahaemolyticus* (sp.) SAs-18, which exhibits good degradation effects against sulfachlorpyridazine, from the topsoil of a heavily polluted area at a pharmaceutical factory wastewater discharge site in Foshan City, Guangdong Province. Ancylobacter SAs-18 (sp.) can be used for the degradation and removal of sulfachlorpyridazine, providing an economical and green method for removing this type of novel organic pollutant.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment, specifically relating to a sulfachlorpyridazine-degrading bacterium and its application. Background Technology

[0002] Sulfonamide antibiotics are broad-spectrum antibacterial agents widely used in agriculture, animal husbandry, and clinical medicine. After use, approximately 50% to 90% of them enter the soil and aquatic environment in their original form or as metabolites, forming persistent residues. These residues not only disrupt the microbial community structure of ecosystems but also induce environmental microorganisms to develop drug-resistant genes, which can then spread through the food chain and threaten human health. Therefore, screening for highly efficient sulfonamide antibiotic-degrading microorganisms has become one of the key research focuses in the field of environmental remediation.

[0003] Microbial degradation is an environmentally friendly technology for treating sulfachlorpyridazine pollution. Currently, several genera of sulfachlorpyridazine-degrading bacteria have been isolated from activated sludge, soil, or aquatic environments, including Acinetobacter (…). Acinetobacter sp.), Pseudomonas (sp.), Pseudomonas ( Pseudomonas sp.) and Bacillus (sp.) Bacillus These strains (e.g., sp.) can transform or mineralize pollutants by breaking the functional groups of sulfachlorpyridazine through their own metabolic enzyme systems.

[0004] Fritillaria cirrhosa is widely distributed in the natural environment. Ancylobacter (sp.) also shows potential in environmental pollution remediation. Current research on *Vibrio parahaemolyticus* mainly focuses on the degradation of halogenated organic matter and environmental adaptability. Existing studies have confirmed the potential of strains. Ancylobacter aquaticus It can grow using 1,2-dichloroethane as the sole carbon source, achieves efficient degradation of halogenated organic compounds by secreting dehalogenases, and exhibits significant environmental resilience.

[0005] However, there are currently no research reports on the degradation of sulfachlorpyridazine by strains of this genus. Given its potential application in environmental pollution control, developing this novel microbial resource of *Tridentobacterium* is of great significance for expanding the degradation pathways of sulfachlorpyridazine. Summary of the Invention

[0006] The purpose of this invention is to provide a sulfachlorpyridazine-degrading bacterium and its applications.

[0007] The sulfachlorpyridazine-degrading bacteria of the present invention are *Tridentibacterium tumefaciens* (…). Ancylobacter sp.) SAs-18, its accession information: depositary institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC), deposit date: November 25, 2025, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, Postcode: 510070, accession number: GDMCCNo.67354.

[0008] The present invention also provides a microbial inoculant containing the above-mentioned Trichoderma SAs-18 or its fermentation broth as an active ingredient.

[0009] Preferably, the microbial agent can be a microbial additive.

[0010] The present invention also provides the application of the above-mentioned Trichoderma SAs-18 or microbial agents in the degradation of sulfachlorpyridazine.

[0011] Preferably, the sulfachlorpyridazine includes sulfachlorpyridazine in various environments, such as soil and water.

[0012] The present invention also provides a method for degrading sulfachlorpyridazine, which involves inoculating the above-mentioned Trichoderma SAs-18 or microbial agent into an environment containing sulfachlorpyridazine, and using Trichoderma SAs-18 to degrade sulfachlorpyridazine.

[0013] This invention isolates a strain of *Tridentium* with good degradation effect on sulfachlorpyridazine from the topsoil of a heavily polluted area at a pharmaceutical factory wastewater discharge site in Foshan City, Guangdong Province. Ancylobacter SAs-18 (sp.) can be used for the degradation and removal of sulfachlorpyridazine, providing an economical and green method for removing this type of novel organic pollutant.

[0014] Ancylobacter sp. SAs-18, its deposit information: depositary institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC), deposit date: November 25, 2025, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, Postcode: 510070, deposit number: GDMCC No. 67354. Attached Figure Description

[0015] Figure 1 This is the colony morphology of strain SAs-18 in LB medium.

[0016] Figure 2 This describes the degradation effect of strain SAs-18 on sulfachlorpyridazine in MSM liquid medium. Detailed Implementation

[0017] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0018] Example 1: Experimental Methods and Results

[0019] 1. Isolation and purification of sulfachlorpyridazine-degrading bacteria SAs-18

[0020] MSM liquid culture medium: 1 L mini-Q water, 9 g K2HPO4·3H2O, 3 g KH2PO4, 0.5 g NaCl, 1 g NH4Cl, 0.25 g MgSO4·7H2O, 0.014 g anhydrous CaCl2, 1 mL trace element solution. Mix all ingredients thoroughly, sterilize at 121℃, and then add 1 mL vitamin stock solution.

[0021] Trace element solution: 0.636 g / L NaHCO3, 0.1 g / L ZnSO4·7H2O, 0.05 g / L CuSO4·5H2O, 0.01 g / L (NH4)6Mo7O 24 ·4H2O, 0.25 g / L CoCl2·6H2O, 0.25 g / L FeSO4·7H2O, 0.15 g / LMnCl2·4H2O.

[0022] Vitamin stock solution: 20 mg / L biotin, 20 mg / L floic acid, 100 mg / L pyridoxine hydrochloride, 50 mg / L riboflavin, 50 mg / L thiamine, 50 mg / L nicotinic acid, 50 mg / L pantothenic acid, 1 mg / L vitamin B12, 50 mg / L p-aminobenzoic acid, 50 mg / L thioctic acid.

[0023] LB liquid medium: 10.0 g peptone, 5.0 g yeast extract, 5.0 g sodium chloride, 1.0 g glucose, 1 L distilled water. Mix all ingredients thoroughly and sterilize before use.

[0024] LB solid medium: 10.0 g peptone, 5.0 g yeast extract, 5.0 g sodium chloride, 1.0 g glucose, 15.0 g agar, 1 L distilled water. Mix all ingredients thoroughly and sterilize before use.

[0025] The topsoil of a heavily polluted area at the wastewater discharge site of a pharmaceutical factory in Foshan City, Guangdong Province, was used as the screening soil sample. Two soil samples, each weighing 15.0 g, were accurately weighed into 50 mL sterile centrifuge tubes. MSM liquid culture medium was added to the 50 mL mark and the tubes were shaken upside down. The tubes were centrifuged at 8000 rpm for 5 min and the supernatant was discarded.

[0026] Two soil samples, totaling 30 g of topsoil, were transferred together to a 250 mL Erlenmeyer flask containing 120 mL of MSM liquid culture medium, bringing the total volume to 150 mL. The target compounds were added to achieve a concentration of 1 mg / L for each target pollutant in the liquid. A control group was also set up, consisting of 30 g of sterilized sludge (intermittent sterilization) and 120 mL of MSM liquid culture medium, with a final target pollutant concentration of 1 mg / L.

[0027] Periodically sample, manually shake well, collect 1 mL of culture medium, centrifuge (12000 rpm, 3 min), pass through a polytetrafluoroethylene membrane into a 2 mL brown vial, and store at -20 ℃ for analysis. Mass spectrometry is used to detect the removal rate of the target analyte; when the removal rate is above 80%, subculturing and enrichment are initiated.

[0028] Each generation was performed in triplicate. In the first generation, 30 mL of the previous generation inoculum was transferred to a 250 mL Erlenmeyer flask containing 120 mL of MSM liquid medium, resulting in a total system volume of 150 mL. The concentration of the target compound was increased stepwise from 1 mg / L, 2 mg / L, 3 mg / L, and finally up to 4 mg / L. When the degradation rate was greater than 80%, the next subculture was performed. For each subculture, the bacterial culture was filtered through a 0.22 μm membrane, and three replicates (30 mL of each membrane) were centrifuged. The membranes were then frozen at -80 °C for DNA extraction.

[0029] Take 100 μL of bacterial culture and add it to a 2 mL centrifuge tube containing 900 μL of sterile water to obtain 10 -1 Bacterial solution, following this method, yielded 10... -2 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 For bacterial suspension, take 100 μL of diluted bacterial suspension and spread it onto LB solid medium containing the target compound.

[0030] Incubate the solid plates upside down in a 30°C incubator until colonies appear. Pile a single colony onto the same solid plate for purification.

[0031] After secondary purification, single bacteria were selected and cultured in LB+ antibiotic liquid medium until turbidity was visible to the naked eye. The bacterial solution was then placed in a preservation tube and glycerol was added to make the final concentration 25%. The bacteria were then preserved in a -80 ℃ freezer, and strain SAs-18 was obtained by screening.

[0032] 2. Morphological and molecular identification of strains

[0033] Using strain SAs-18, screened in Example 1, as the research object, it was inoculated onto LB solid medium and incubated upside down in a 30 ℃ incubator until colonies appeared. The colony morphology on the plates was then observed. The results are as follows: Figure 1 As shown, the colonies of the strain SAs-18, when cultured on nutrient agar medium, are round, smooth, pale yellow, and uniform in shape.

[0034] The bacterial strain was sent to Beijing Liuhe BGI Genomics Co., Ltd. for 16S rDNA identification. Sequence amplification was performed using universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3'), SEQ ID NO.2, and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.3), with total bacterial DNA as a template. The PCR reaction program was as follows: denaturation at 96 ℃ for 15 min, 96 ℃ for 30 s, 56 ℃ for 30 s, and 72 ℃ for 1 min, for a total of 35 cycles; storage at 72 ℃ for 5 min and at 4 ℃. The obtained 16S rDNA sequence (its nucleotide sequence is shown in SEQ ID NO.1) was entered into GenBank for BLAST homology alignment. The alignment revealed that strain SAs-18 is similar to... Ancylobacter The strain SAs-18 is most closely related to strain Alpha-13, with a homology exceeding 99%. Based on morphological, physiological, and biochemical indicators, as well as 16S rDNA gene sequence analysis, strain SAs-18 was preliminarily identified as... Ancylobacter sp., named Fritillaria ( Ancylobacter sp.) SAs-18, its deposit information: depositary institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC), deposit date: November 25, 2025, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, Postcode: 510070, deposit number: GDMCC No. 67354.

[0035] The aforementioned Fritillaria ( Ancylobacter The 16S rDNA sequence of sp.)SAs-18 is shown in SEQ ID NO:1 below.

[0036] 3. Degradation of sulfachlorpyridazine by strain SAs-18 in MSM liquid medium

[0037] The aforementioned Fritillaria ( Ancylobacter Application of SAs-18 (sp.) in the biological treatment of antibiotic-contaminated wastewater. In particular, it can be used to degrade sulfachlorpyridazine in aquaculture wastewater. This bacterium can degrade sulfachlorpyridazine under aerobic conditions.

[0038] MSM liquid culture medium was prepared, sterilized, and cooled to room temperature. Under aseptic conditions, it was dispensed into 250 mL Erlenmeyer flasks, and the target sulfachlorpyridazine was added to achieve an initial concentration of 1 mg / L. The experiment was divided into two groups: a sterile experimental group, inoculated with *Trèsrus repens* SAs-18 into the MSM liquid culture medium and mixed thoroughly; and a sterile control group, without inoculation with *Trèsrus repens* SAs-18, wrapped in aluminum foil to protect from light, and cultured in a shaker at 30 ℃ and 150 rpm. Both experimental and control groups were replicated in triplicate. The experimental period was 7 days, with sampling frequency of once daily. The residual amount of sulfachlorpyridazine in the culture medium was determined by liquid chromatography to analyze the degradation ability of *Trèsrus repens* SAs-18 on sulfachlorpyridazine. The results are as follows: Figure 2 As shown in the figure, in the treatment group, *Tridentium flexneri* SAs-18 had a significant degradation effect on sulfachlorpyridazine, with a removal rate of 91.0% after 7 days; in the sterile blank control group, the residual amount of sulfachlorpyridazine did not change significantly.

[0039] The strain of this invention has the ability to efficiently degrade sulfachlorpyridazine. In MSM liquid medium, the degradation rate of 1 mg / L sulfachlorpyridazine (SCP) reached 91% after 7 days.

Claims

1. Fritillaria ( Ancylobacter sp.) SAs-18, accession number: GDMCC No.67354.

2. A microbial inoculant, characterized in that, It contains the Bacillus SAs-18 or its fermentation broth as the active ingredient as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The aforementioned microbial agent is a microbial additive.

4. The use of the *Tridentium rubrum* SAs-18 of claim 1 or the microbial agent of claim 2 in the degradation of sulfachlorpyridazine.

5. The application according to claim 4, characterized in that, The sulfachlorpyridazine mentioned is sulfachlorpyridazine found in soil and water environments.

6. A method for degrading sulfachlorpyridazine, characterized in that, The method involves inoculating the *Tridentiformis* SAs-18 of claim 1 or the microbial agent of claim 2 into an environment containing sulfachlorpyridazine, and using *Tridentiformis* SAs-18 to degrade sulfachlorpyridazine.

7. The method according to claim 6, characterized in that, The sulfachlorpyridazine mentioned is sulfachlorpyridazine found in soil and water environments.

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