Dehalococcoides sp. and application thereof in degrading halogenated aromatic hydrocarbon compounds

Abstract

The application belongs to the field of microorganisms, and particularly relates to a strain of dehalococcoides and application thereof in degrading halogenated aromatic hydrocarbon compounds. The dehalococcoides DCF strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on March 18, 2026, and the address of the CGMCC is No. 1, Xibahe Road, Yard 3, Chaoyang District, Beijing, and the postal code is 100101, and the Institute of Microbiology, Chinese Academy of Sciences; the preservation number is CGMCC No. 46936. The degradation capacity of the DCF strain provides important strain resources for in-situ remediation of halogenated aromatic hydrocarbon contaminated sites.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a dehalogenated monoclonal bacterium and its application in the degradation of halogenated aromatic hydrocarbon compounds. Background Technology

[0002] Halogenated aromatic hydrocarbons are a class of compounds formed when one or more hydrogen atoms in aromatic hydrocarbons and their derivatives are replaced by halogen atoms. They mainly include chlorobenzene, chlorophenol, chlorophenoxyacid, chloropolycyclic aromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs). Diclofenac, a typical halogenated aromatic hydrocarbon and nonsteroidal anti-inflammatory drug (NSAID), possesses analgesic, anti-inflammatory, and antipyretic properties and is clinically used to treat various acute and chronic pain and inflammatory diseases. Approximately 6-15% of ingested diclofenac residues are excreted directly in urine and subsequently enter wastewater collection systems. In conventional wastewater treatment processes, diclofenac exhibits poor biodegradability and limited adsorption removal, resulting in typically low removal efficiency (30-70%). Therefore, wastewater treatment plant effluent is a major source of diclofenac in the environment.

[0003] Organohalogen-respiring bacteria (OHRB)-mediated reductive dehalogenation is one of the main pathways for microbial degradation and transformation of organohalides in anaerobic environments. These microorganisms use organohalides as electron acceptors and hydrogen, formic acid, or acetic acid as electron donors. Through reductive dehalogenase catalysis, they obtain the energy required for growth by replacing halogen groups with hydrogen atoms in a reductive dehalogenation reaction. OHRBs play an important role in the removal of organohalides from anaerobic environments such as groundwater and sediments, such as *Dehalogenated Coccidia* (…). Dehalococcoides This technology can completely dechlorinate the priority pollutants trichloroethylene and monochloroethylene, converting them into non-toxic and harmless ethylene. Commercially available bacterial agents such as KB-1 and SDC-9, developed with *Dehalogenated Coccidia* and *Dehalogenated Monoclonal* strains as core functional strains, have been widely used in the remediation of groundwater contaminated with organochlorine. *Dehalogenated Monoclonal* is a class of organohalogen-respiring bacteria discovered in recent years, representing a new genus in the class *Dehalogenated Coccidia* of the phylum *Vibrio*, and was among the first to be discovered... Dehalogenimonas lykanthroporepellens Strains such as BL-DC-9 undergo organohalogen respiration via dihalogen elimination reactions. New members of this genus discovered in recent years (e.g., Dehalogenimonas etheniformans GP strains can achieve dechlorination via hydrogenation and degrade various chlorinated olefins, including vinyl chloride. Many pharmaceutical and personal care product compounds contain halogenated aromatic hydrocarbons, but research on the anaerobic microbial transformation of these compounds is limited. Studies on the degradation of the nonsteroidal anti-inflammatory drug diclofenac via the organohalogen respiratory pathway have not been reported; in particular, no organohalogen respiratory bacteria capable of degrading diclofenac have been found in the genus *Dehalogenomonas*. Currently, domestic and international research teams generally use activated sludge or wastewater from sewage treatment plants as inoculum sources (mixed bacterial communities) to study the degradation of diclofenac, but diclofenac exhibits difficult-to-degrade characteristics, with degradation conversion rates all below 50% (0%-45.8%). Summary of the Invention

[0004] The purpose of this invention is to provide a dehalogenated monoclonal bacterium and its application in the degradation of halogenated aromatic hydrocarbon compounds.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A strain of dehalogenated monoclonal bacteria ( Dehalogenimonas (sp.), Dehalogenated Monoclonal strain DCF, which was deposited on March 18, 2026 at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode: 100101; accession number: CGMCC No. 46936.

[0006] The DCF strain is a Gram-negative bacterium. Based on the 16S rRNA gene sequence similarity comparison, it has a species similarity of 99.22% with the GP strain of the genus Dehalomonas. Therefore, the DCF strain is identified as belonging to the genus Dehalomonas.

[0007] An application of the aforementioned dehalogenated monoclonal bacteria, specifically the application of the DCF strain in the degradation of halogenated aromatic hydrocarbons.

[0008] The halogenated aromatic hydrocarbon compound is diclofenac.

[0009] A bacterial agent for degrading halogenated aromatic hydrocarbons, the bacterial agent containing the aforementioned dehalogenated monoclonal bacteria DCF strain.

[0010] The bacterial agent is one or more of the strain's culture, bacterial suspension, or concentrate.

[0011] The application of a microbial agent for degrading halogenated aromatic hydrocarbon compounds, wherein the microbial agent is used in the degradation of halogenated aromatic hydrocarbon compounds.

[0012] A method for degrading halogenated aromatic hydrocarbons involves inoculating the described dehalogenated monoclonal bacteria (DCF strain) or the bacterial agent into an anaerobic environment to be treated, and adding a carbon source and an electron donor, thereby degrading the halogenated aromatic hydrocarbons in the environment.

[0013] The carbon source is acetate, with a final concentration of 5 mM in the system, and the electron donor is hydrogen gas; the halogenated aromatic hydrocarbon compound is diclofenac.

[0014] Furthermore, the strain was cultured in an anaerobic inorganic salt medium with added carbon source, electron donor, and electron acceptor, and the organic halogenated dehalogenated monoclonal bacteria (DCF) strain was inoculated and cultured at pH 7.2, 30°C, and in the dark for 160-180 days. The carbon source and electron donor were 5 mM acetate and hydrogen, respectively, with an inoculum size of 3%. The resulting culture was centrifuged, the precipitate was collected, and resuspended to obtain a bacterial suspension. The culture was then concentrated to obtain a concentrate.

[0015] Advantages of this invention: The dehalogenated monoclonal bacteria (DCF) strain obtained in this invention is a type of organohalogenated respiratory bacteria that degrades halogenated aromatic hydrocarbons, screened from polluted river sediment.

[0016] Based on the 16S rRNA gene sequence similarity comparison, strain DCF showed a species similarity of 99.22% with strain GP of the genus Dehalomonas. Therefore, strain DCF was identified as belonging to the genus Dehalomonas.

[0017] Under growth conditions of 30°C and pH 7.2, the DCF strain can degrade diclofenac into 2-(2-((2-chlorophenyl)amino)phenyl)acetic acid (monoclofenac), and finally into the halogen-free product 2-((2,6-dichlorophenyl)amino)phenylacetic acid (anilinephenylacetic acid). The degradation ability of the DCF strain provides an important microbial resource for the in-situ remediation of sites contaminated with halogenated aromatic hydrocarbons. Attached Figure Description

[0018] Figure 1 The image shows the cell morphology of the DCF strain under a scanning electron microscope, as provided in an embodiment of the present invention.

[0019] Figure 2 The sequencing chromatogram of the DCF pure culture strain Sanger provided in this embodiment of the invention.

[0020] Figure 3 A phylogenetic tree was constructed by homology comparison of the 16S rRNA gene sequences of the DCF strain provided in this embodiment of the invention and representative strains reported previously.

[0021] Figure 4 The DCF strain and dehalogenated monocytogenes GP provided in the embodiments of the present invention T Degradation curves of diclofenac by the strain.

[0022] A strain of dehalogenated monoclonal bacteria ( Dehalogenimonas(sp.), Dehalogenated Monoclonal strain DCF, which was deposited on March 18, 2026 at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode: 100101; accession number: CGMCC No. 46936. Detailed Implementation

[0023] The technical solution of the present invention will be further explained below with reference to specific embodiments, but it should not be construed as a limitation of the present invention.

[0024] Example 1: Isolation, purification and identification of strains (1) Preparation of basal culture medium: The anaerobic inorganic salt culture medium consisted of: NaCl 1.0 g / L, MgCl₂·6H₂O 0.5 g / L, KH₂PO₄ 0.2 g / L, NH₄Cl 0.3 g / L, KCl 0.3 g / L, CaCl₂·2H₂O 0.015 g / L, FeCl₂·4H₂O 1.5 mg / L, CoCl₂·6H₂O 190 μg / L, MnCl₂·4H₂O 100 μg / L, ZnCl₂ 70 μg / L, H₃BO₃ 6 μg / L, Na₂MoO₄·2H₂O 36 μg / L, NiCl₂·6H₂O 24 μg / L, CuCl₂·2H₂O 2 μg / L, Na₂SeO₃·5H₂O 6 μg / L, and Na₂WO₄·2H₂O 8 μg / L. The culture medium was prepared with 0.025% (w / v) resazu indicator, 24 mg / L L-cysteine, 38.5 mg / L dithiothreitol, and 2.52 g / L (30 mM) NaHCO3. The pH was adjusted to 7.2-7.3. After autoclaving at 121℃ for 15 minutes, a multivitamin was added. The final concentrations of various vitamins in the culture medium were as follows: biotin 20 μg / L, folic acid 20 μg / L, pyridoxine hydrochloride 100 μg / L, riboflavin 50 μg / L, thiamine 50 μg / L, pantothenic acid 50 μg / L, nicotinic acid 50 μg / L, vitamin B12 50 μg / L, para-aminobenzoic acid 50 μg / L, and lipoic acid 50 μg / L.

[0025] (2) Enrichment of anaerobic bacteria that degrade halogenated aromatic hydrocarbons: Dispense 80 mL of the anaerobic inorganic salt culture medium described in step (1) above into a 100 mL serum bottle, add 5 mM sodium acetate as a carbon source and 5 mL of hydrogen as an electron donor, and simultaneously add 100 µM diclofenac as an electron acceptor. The headspace gas is N2 / CO2 (80 / 20, v / v). In an anaerobic glove box, inoculate 3 mL of sediment mud suspension (collected from Xihe, Shenyang, Liaoning) and seal the serum bottle with a rubber stopper and aluminum cap to establish an enrichment culture system. Incubate statically at pH 7.2, 30℃, and in the dark. Monitor the degradation process of diclofenac using high performance liquid chromatography. After diclofenac is converted to monoclofenac, a small amount of monoclofenac is further converted to aniline phenylacetic acid. Repeat the transfer to the same composition culture medium 5 times with a 3% inoculum.

[0026] (3) Isolation of anaerobic bacteria that degrade halogenated aromatic hydrocarbons: Dispense 9 mL of the inorganic culture medium described in step (1) into a 20 mL culture flask. The headspace gas is N2 / CO2 (80 / 20, v / v). After sealing with a rubber stopper and aluminum cap, add 100 µM diclofenac via a microsyringe. Transfer 1 mL of the enriched culture of the anaerobic bacteria that degrade halogenated aromatic compounds described above from the 120 mL serum bottle described in step (2) into the culture flask to establish a 10-year culture. -1 Dilute bottles, and so on, repeat the above 10-fold serial dilution operation until a 10-fold serial dilution is established. -10 Dilution vials. The degradation and transformation process is monitored using liquid chromatography, and high dilution vials (e.g., 10⁻⁶) are prepared. -8 10 -9 10 -10 After diclofenac in the culture medium is completely degraded to monoclofenac, about 2.5 mL of the culture medium is transferred to the inorganic salt culture medium described in (1). The same carbon source, electron donor and electron acceptor as the culture system in (2) are added, and the culture is incubated at 30°C in the dark. The monoclofenac production process is monitored by liquid chromatography, and the diclofenac degrading strain is isolated and purified.

[0027] (4) Identification of strains: The haloaromatic hydrocarbon degrading strains obtained through the above process are approximately spherical. Figure 1 The genome was extracted using a soil genomic DNA extraction kit. PCR amplification was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3' (sequence 1)) and 1492R (5'-GGTTACCTTGTTACGACTT-3' (sequence 2)) for the bacterial 16S rRNA gene. The amplified products were submitted to a sequencing company for sequencing. The sequencing chromatogram showed a single peak without any impurities, indicating a pure culture strain. Figure 2 The 16S rRNA gene fragment obtained by PCR amplification was 1170 bp, with a G+C mol% of 53.5%.

[0028] The 16S rRNA gene sequence (5'-3') is as follows:

[0029] BLAST search results for the 16S rRNA gene sequence of this strain showed that it shared 99.22% species homology with strains from the genus *Dehalomonas* GP. Figure 3 This strain was classified under the genus *Dehalogenated Monoclonal* and named strain DCF (…). Dehalogenimonas The sp. strain DCF is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on March 18, 2026, 100101, China. Accession number: CGMCC No. 46936.

[0030] Example 2: Identification of the degradation performance of diclofenac by strain DCF In the 80 mL of Example 1 described in step (1), 5 mM sodium acetate was added as a carbon source and 5 mL of hydrogen gas as an electron donor to the anaerobic inorganic salt basal medium, and 100 µM diclofenac was added as an electron acceptor. 3% (v / v) of pure DCF bacteria was inoculated as the experimental group, and dehalogenated GP bacteria were used. T The strain (see the literature *Dehalogenimonas etheniformans sp. nov., a formate-oxidizing,organohalide-respiring bacterium isolated from grape pomace*, which is the type strain of this genus) served as a control group and was cultured statically at pH 7.2, 30°C, and in the dark. The conversion of diclofenac was monitored using an Agilent 1260Ⅱ series high-performance liquid chromatography (HPLC) column with a tandem diode array variable wavelength detector. A reversed-phase C18 column (Agilent Technologies, 4.6 mm diameter × 250 mm length, 5 µm particle size) was used, with a column temperature of 35°C. Elution was performed isocratically with 2% acetic acid:100% acetonitrile (40:60 v / v) at a flow rate of 1.0 mL / min for 8 minutes, and detection was performed at 276 nm. The quantitative linear range for both diclofenac and monoclofenac was 1.0–10.0 µM, with a detection limit of 0.3 µM. The conversion of diclofenac was detected based on the measured values, and the degradation products were quantitatively analyzed.

[0031] See Figure 4 Analysis showed that the DCF strain could progressively degrade 100 µM diclofenac over 90 days, first converting it to monoclofenac, and then further converting some of the monoclofenac into aniline phenylacetic acid. In contrast, the control group GP... T The strain cannot degrade or transform diclofenac.

[0032] Currently, there are no reports of pure cultured microorganisms that anaerobically degrade diclofenac. Domestic and foreign research teams have studied the degradation of diclofenac using activated sludge or wastewater from sewage treatment plants as inoculum sources (mixed microbial communities). Diclofenac has shown that it is difficult to degrade, and the degradation conversion rate is less than 50% (0%-45.8%).

[0033] The bacterial agent was prepared according to the above description. A carbon source, electron donor, and electron acceptor were added to an inorganic salt culture medium. The *Dehalogenated Monoclonalella* (DCF) strain was inoculated and cultured statically at pH 7.2, 30°C, and in the dark for 100 days. The carbon source and electron donor were acetate and hydrogen gas, respectively. 5 mM sodium acetate, 5 mL hydrogen gas, and 100 µM diclofenac were added to every 80 mL of inorganic salt culture medium, and the DCF strain was inoculated at a rate of 3% (v / v). The resulting culture was centrifuged, the precipitate was collected, and resuspended to obtain a bacterial suspension. The culture was then concentrated to obtain a concentrate, which is the bacterial agent. Applying this to the environment or soil shows promising application prospects for the bioremediation of sites contaminated with halogenated aromatic hydrocarbons.

[0034] The embodiments described above are preferred application examples of this invention, but do not constitute any limitation on this invention. In practical applications, without departing from the scope of the technical solution of this invention, some modifications or alterations can be made to the disclosed technical content to create equivalent embodiments.

Claims

1. A strain of dehalogenated monoclonal bacteria ( Dehalogenimonas sp.), characterized by: The DCF strain of *Dehalogenated Monoclonalella* was deposited on March 18, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China; accession number: CGMCC No. 46936.

2. The application of the dehalogenated monoclonal bacteria according to claim 1, characterized in that: Application of the dehalogenated monoclonal bacteria (DCF) strain in the degradation of halogenated aromatic hydrocarbons.

3. The application of the dehalogenated monoclonal bacteria according to claim 2, characterized in that: The halogenated aromatic hydrocarbon compound is diclofenac.

4. A bacterial agent for degrading halogenated aromatic hydrocarbons, characterized in that: The bacterial agent contains the dehalogenated monoclonal strain DCF as described in claim 1.

5. The microbial agent for degrading halogenated aromatic hydrocarbons according to claim 4, characterized in that: The bacterial agent is one or more of the strain's culture, bacterial suspension, or concentrate.

6. The application of the microbial agent for degrading halogenated aromatic hydrocarbons as described in claim 5, characterized in that: The application of the bacterial agent in the degradation of halogenated aromatic hydrocarbons.

7. A method for degrading halogenated aromatic hydrocarbons, characterized in that: By inoculating the dehalogenated monoclonal bacteria DCF strain of claim 1 or the bacterial agent for degrading halogenated aromatic hydrocarbons of claim 4 into the anaerobic environment to be treated, and adding a carbon source and an electron donor, the halogenated aromatic hydrocarbons in the environment can be degraded.

8. The method for degrading halogenated aromatic hydrocarbons according to claim 7, characterized in that: The carbon source is acetate, with a final concentration of 5 mM in the system, and the electron donor is hydrogen gas; the halogenated aromatic hydrocarbon compound is diclofenac.

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