Dehalobacterium with dichloromethane anaerobic degradation ability and application thereof

Abstract

This invention discloses a field of environmental pollution remediation, and more specifically, relates to a dehalobacterium strain capable of anaerobic degradation of dichloromethane and its applications. The strain is a dehalobacterium strain DCM, which was deposited on November 30, 2021, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 24007. The novel dehalobacterium strain DCM provided by this invention was isolated and purified from contaminated river sediments. The strain is Gram-positive, rod-shaped, with a smooth surface, a cell length of 1-2 μm, and a diameter of 0.3-0.5 μm. It can completely convert 160 μmol of dichloromethane into formate and acetate within 7 days in an anaerobic inorganic salt medium, with a degradation rate as high as 32 μmol / L. ‑1 This indicates that the strain can efficiently degrade dichloromethane pollutants and has broad application prospects in environmental wastewater treatment and environmental remediation.

Description

Technical Field

[0001] This invention discloses the field of environmental pollution remediation, and more specifically, relates to a dehalogenated bacillus with the ability to anaerobicly degrade dichloromethane and its applications. Background Technology

[0002] Dichloromethane (DCM) is an important volatile halogenated hydrocarbon widely used in industrial production, primarily in adhesives, pesticides, metal degreasers, and paint removers. With accelerating urbanization and industrialization, the demand for DCM is increasing daily. This extensive use leads to its entry into the environment through wastewater, gases, and solid waste, particularly into groundwater, soil, and the atmosphere. Environmental concentrations of DCM are often high in industrial areas, landfills, and around chemical plants. This compound is highly volatile, highly water-soluble, and has low adsorption capacity, making it highly mobile in the environment. Furthermore, DCM has potential toxicity to humans and ecosystems. Short-term exposure can cause neurotoxicity, respiratory irritation, and skin damage, while long-term exposure may lead to hepatotoxicity and carcinogenic risks. Therefore, the International Agency for Research on Cancer (IARC) classifies it as a possible human carcinogen (Group 2B). DCM is difficult to degrade naturally in the environment and exhibits environmental persistence. In recent years, with the increasing awareness of environmental protection and the implementation of relevant regulations, environmental control of dichloromethane during its production and use has received increasing attention. Currently, dichloromethane has been included in the first batch of the "List of Key New Pollutants under Control (2023 Edition)" issued by the Ministry of Ecology and Environment of my country, and has also been included in the list of toxic and hazardous air pollutants and the list of toxic and hazardous water pollutants, making it a new pollutant that requires key control.

[0003] Traditional physicochemical remediation technologies are costly and prone to secondary pollution. In contrast, bioremediation technologies utilize microorganisms to convert dichloromethane into low-toxicity or non-toxic substances, offering a green, economical, and efficient solution. To date, the aerobic dichloromethane-degrading bacteria reported mainly belong to genera such as *Hyphomicrobium*, *Albibacter*, *Ancylobacter*, *Bacillus*, *Gottschalkia*, *Lysinobacillus*, *Paracoccus*, *Methylophilus*, *Methylopila*, and *Methylobacterium*. However, even in microaerobic and anoxic environments, large amounts of dichloromethane pollutants are present, but anaerobic microbial populations capable of metabolizing dichloromethane are very few. For example, the *Dehalobacterium formicoaceticum* DMC strain reported in 1996 is currently the only pure culture capable of anaerobicly degrading dichloromethane. As early as 2012, cultures of microorganisms highly enriched with *Dehalobacter* were thought to be capable of anaerobic metabolism of dichloromethane. However, due to limitations in isolation techniques, pure cultures of this *Dehalobacter* have not yet been obtained. This directly leads to problems such as low efficiency in anaerobic remediation of dichloromethane, insufficient adaptability, limited technological development, increased ecological risks, and over-reliance on other remediation methods. Therefore, obtaining a biomaterial capable of anaerobic degradation of dichloromethane, especially a novel *Dehalobacter*, represents a major breakthrough in the field of environmental bioremediation. Summary of the Invention

[0004] In view of the above, the purpose of this invention is to provide a dehalogenated bacillus with the ability to anaerobicly degrade dichloromethane and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A dehalobacterium strain capable of anaerobic degradation of dichloromethane, strain DCM, was deposited on November 30, 2021, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 24007.

[0007] The bacteria are rod-shaped, Gram-positive, 1-2 μm in length, and 0.3-0.5 μm in diameter.

[0008] An application of the aforementioned dehalogenated Bacillus strain in the degradation of dichloromethane contaminants in the environment.

[0009] The application of the strain in degrading dichloromethane pollutants in the environment under anaerobic conditions.

[0010] The environment is water, sediment, or soil.

[0011] An agent for degrading dichloromethane contaminants, the agent comprising the aforementioned dehalogenated bacillus strain DCM.

[0012] The preparation contains cell cultures of the strain, cell culture concentrates, cell culture filtrates, or culture suspensions.

[0013] A method for degrading dichloromethane pollutants involves inoculating the bacterial strain or the bacterial agent into an environmental sample to be treated, adding sodium sulfide at a final concentration of 0.05 mM as an oxygen scavenger to the environmental sample, thereby achieving anaerobic degradation of dichloromethane in the environmental sample.

[0014] The inoculation amount of the strain or agent is 0.1% (w / v).

[0015] The present invention has the following advantages and effects compared with the prior art:

[0016] This invention provides a dehalobacterial bacterium strain, *Dehalobacter sp.*DCM, capable of degrading dichloromethane. Quantitative analysis of dichloromethane using gas chromatography revealed that the strain possesses the ability to rapidly consume DCM. Qualitative and quantitative analysis of the degradation products of DCM using liquid chromatography showed that the degradation products are acetic acid and formic acid, demonstrating that this bacterium has the ability to efficiently degrade dichloromethane pollutants into non-toxic small-molecule organic acids, and has the potential to be applied to groundwater, soil, and other environments for the biodegradation of dichloromethane. Attached Figure Description

[0017] Figure 1 This is a comparison chart of the degradation rates of dichloromethane by different single colonies in the embodiments of the present invention;

[0018] Figure 2 This is a scanning electron microscope image of the novel dehalobacter sp. DCM strain isolated in the embodiments of the present invention;

[0019] Figure 3 This is a phylogenetic diagram of the novel dehalobacter sp. DCM strain of the present invention;

[0020] Figure 4 This is a degradation curve of dichloromethane to acetic acid and formic acid by the novel dehalobacter sp. DCM strain in this embodiment of the invention.

[0021] Figure 5This invention demonstrates the degradation of dichloromethane by the novel dehalobacter sp. DCM strain at different culture temperatures. Detailed implementation method:

[0022] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings. It should be noted that the specific embodiments described herein are only for illustration and explanation of the present invention and are not limited thereto.

[0023] Unless otherwise specified, the following implementation schemes generally follow standard testing conditions or the methods recommended by the reagent company. Unless otherwise specified, all materials, reagents, and equipment used are conventional materials, reagents, and equipment in this technical field and are commercially available.

[0024] Example 1. Isolation and purification of strains

[0025] (1) Sample collection

[0026] Sediment samples were collected from the Xihe River sewage outlet in Shenyang City, Liaoning Province (latitude: 41°39′46″, longitude: 123°6′20″). The sediment was dug deep into the riverbed using a high-temperature sterilized shovel. The collected sediment was quickly transferred to a 2L sterile collection bottle, and river water was added to remove any air remaining in the headspace of the bottle. The bottle was then sealed with a blue rubber stopper. The collected samples were promptly stored in a 4°C refrigerator, and microbial screening was conducted within one month.

[0027] (2) Construction of artificial micro-ecosystems

[0028] First, prepare the anaerobic inorganic salt liquid culture medium according to Table 1. Heat and boil it under nitrogen blowing conditions, and add L-cysteine ​​24 mg / L, Na2S·9H2O 48 mg / L and dithiothreitol (DTT, 77 mg / L) as reducing agents to remove oxygen from the culture medium. Adjust the pH of the culture medium to 7.2 with CO2, and sterilize it at 121℃ for 30 min. Take 2g of the fresh sediment collected in step (1) above and inoculate it into a culture flask containing 10 μL of dichloromethane, 0.1 mL of compound vitamins and trace elements (Table 1) to prepare an artificial micro-ecosystem with dichloromethane as the sole carbon source. Place it in a 30℃ biochemical incubator in the dark and let it stand still.

[0029] (3) Enrichment and isolation of dichloromethane-degrading bacteria

[0030] After 30 days of culture, 3 mL of culture medium was used as seed medium and transferred to a new 100 mL anaerobic inorganic salt liquid medium according to the inoculum amount. The enriched culture was then cultured for 11 cycles under the same conditions and subculture method as in step (2) above to obtain an enriched culture with dichloromethane degradation ability. 1 mL of suspension was taken from the enriched medium after the 11th subculture and diluted with sterile anaerobic inorganic salt medium to a concentration of 10 mL. -1 Up to 10 -10 Serial dilution of bacterial culture, 10 -4 Up to 10 -10 The diluted solution was transferred to an inorganic salt semi-solid culture medium with dichloromethane as the sole carbon source, with three replicates for each concentration. The culture was placed in an incubator at 30°C and the colony morphology was observed periodically.

[0031] Table 1. Composition and content of anaerobic inorganic salt liquid culture medium, trace elements, and complex microorganisms used in this invention.

[0032]

[0033]

[0034] Except for containing 1% (w / v) low gel temperature agar (Sigma-Aldrich, Germany, catalog number: A9045), the inorganic salt semi-solid culture medium has the same components and preparation method as the anaerobic inorganic salt liquid culture medium.

[0035] After half a month of cultivation, single colonies of different morphologies were picked and cultured in 100 mL anaerobic inorganic salt liquid medium containing 10 μL of dichloromethane, and the dichloromethane degradation rate was determined. One mL of the bacterial culture of the strain with the highest degradation rate was selected, and the above serial dilution purification steps were repeated three times. The resulting pure cultures capable of degrading dichloromethane were stored at 4°C under numbered labels.

[0036] Degradation rate determination: During the biodegradation of dichloromethane, samples were taken periodically and the degradation rate was determined using gas chromatography-flame ionization detector. Degradation rate = (initial concentration - final concentration) / number of days of incubation.

[0037] Determination of dichloromethane by gas chromatography-flame ionization detector (GC-FID): 1 mL of dichloromethane degradation culture medium was transferred to a 20 mL headspace vial and quickly sealed with a cap gasket. The headspace vial containing the sample was then placed on the injection plate of the gas chromatograph. Instrument parameters were as follows: column (DB-624, 60 m × 0.32 mm × 1.8 μm); split ratio at the injection port was 50:1; injection port temperature was 200 °C; column oven temperature program was 60 °C for 2 min, then increased to 200 °C at a rate of 25 °C / min; carrier gas was 99.999% pure helium at a flow rate of 3 mL / min; fuel gas and oxidizer were hydrogen and synthetic air (99.999% pure), at flow rates of 30 mL / min and 350 mL / min, respectively; detector temperature was 300 °C; make-up gas was nitrogen at a flow rate of 25 mL / min.

[0038] The results showed that eight single colonies of different morphologies were picked from the semi-solid culture medium and named strains A1-A8. A1, A3, and A7 did not exhibit degradative activity against dichloromethane, while A2, A4, A5, A6, and A8 consumed dichloromethane at different rates, with A6 showing the highest degradation rate. Figure 1 ).

[0039] Example 2. Identification of the strain

[0040] (1) Morphological identification:

[0041] After culturing the A6 strain obtained in Example 1 in a semi-solid medium for half a month, the colonies had neat edges, smooth surfaces, and were opaque milky white in color, with a colony diameter of approximately 0.5-1.0 mm. Electron scanning microscopy, using conventional bacterial physiological and biochemical identification methods, showed that the isolated strain was Gram-positive, with rod-shaped cells, no flagella, a cell length of 1-2 μm, and a diameter of 0.3-0.5 μm. Figure 2 ).

[0042] (2) Molecular identification:

[0043] DNA was extracted from the pure culture of the strain obtained in Example 1 using a soil genomic DNA extraction kit. Then, PCR amplification and sequencing were performed using universal primers 27F and 1492E for the 16S rRNA gene to obtain the 16S rRNA gene sequence (SEQ.ID.NO.1). The obtained 16S rRNA gene sequence was entered into the Blastn program of the National Center for Biotechnology Information (NCBI) for comparison and analysis with all nucleotide sequences in the database. A phylogenetic tree was constructed using the 16S rRNA gene sequence. Figure 3The study found that the 16S rRNA gene fragment of the isolated strain was 1617 bp, with a homology of over 95.0% with species of the genus Dehalobacter, and the highest homology of 97.3% with Dehalobacter sp.TCP1, which degrades trichlorophenol.

[0044] Based on the physiological and biochemical characteristics and 16S rRNA gene sequence results described above, the strain isolated in this invention should be classified as a new variant of the genus Dehalobacter, named Dehalobacter sp. (Dehalobacter strain DCM).

[0045] The dehalogenated Bacillus strain DCM was deposited on November 30, 2021, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 24007.

[0046] The 16S rRNA gene sequence of the dehalogenated Bacillus strain DCM is as follows:

[0047] ACACATGCGAGTCGAACGGTCCGATACCTAACACCGAGTGCTTACGT

[0048] GTAAACAGCAAGCATCTAAAGCGAGTGCGCGAACGAAGAGAGCGCA

[0049] CCACGCTATTAAAAAGTGACTGACACATAATGCAGAAGAAAACTTTT

[0050] TAGTAGGTGGGAGCGAACGAAGAGAGCGCACCACGCAAATAAAGCT

[0051] TGCTAACACATGCAATGAGCATTGGGTGTTAGGTAGAGGATAGTGGC

[0052] GAACGGGTGAGTAACGCGTGGGTAACCTGCCCTTAAGACCGGGACA

[0053] ACAGCTGGAAACGGCTGCTAATACCGGATGATTTTCCTTGAAGGCAT

[0054] CTTCGAGGAAGTAAAGCTGGCCTCTGAATATGCTAGCGCTTAGGGAT

[0055] GGACCCGCGTCTGATTAGCTAGTTGGTGGGGTAATGGCCTACCAAGG

[0056] CGACGATCAGTAGCCGGCCTGAGAGGGTAAACGGCCACACTGGGAC

[0057] TGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTT

[0058] CCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGTATGAAGA

[0059] AGGCCTTCGGGTTGTAAAATACTGTTGTTAGGGAAGAACTTTAGAGG

[0060] TGTGAATAATGCCTTTAATTGACGGTACCTAACGAGGAAGCCCCGGC

[0061] TAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGT

[0062] CCGGAATCATTGGGCGTAAAGGGCGCGTAGGCGGCTATATAAGTCTG

[0063] ATGTGAAAGTGCGGAGCTTAACTCCGTAAAGCATTGGAAACTGTATG

[0064] GCTTGAGGACAGGAGAGGAAAGTGGAATTCCACGTGTAGCGGTGAA

[0065] ATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTTTCTG

[0066] GACTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCGAACAGGA

[0067] TTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTA

[0068] GAGGGTATCGACCCCTTCTGTGCCGCAGTTAACACAATAAGCACTCC

[0069] GCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGG

[0070] GGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGACGCAACGCG

[0071] AAGAACCTTACCAAGGCTTGACATCCATAGAATCCTTAAGAGATTAG

[0072] GGAGTGCCCTTCGGGGAACTATGAGACAGGTGGTGCATGGTTGTCGT

[0073] CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC

[0074] CCCTATATTTAGTTGCTAACAGGTAAAGCTGAGAACTCTAGATAGAC

[0075] TGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCA

[0076] TGCCCCTTATGTCTTGGGCTACACACGTGCTACAATGGACGGTACAG

[0077] ACGGAAGCGAAGCCGCGAGGTGAAGCAAATCCGAGAAAGCCGTTCT

[0078] CAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATCGC

[0079] TAGTAATCGCAGGTCAGCACACTGCGGTGAATACGTTCCCGGGCCTT

[0080] GTACACACCGCCCGTCACACCACGAAAGTTTGCAACACCCGAAGCCG

[0081] GTGGGGTAACCGTAAGGAGCCAGCCGTCGAA

[0082] Example 3. Degradation performance of the dehalogenating bacterium strain DCM on dichloromethane

[0083] The degradation performance of dichloromethane was studied by investigating the degradation curve, maximum substrate content, and optimal degradation temperature of dichloromethane by the dehalogenated Bacillus strain DCM.

[0084] The Dehalobacter sp. strain DCM culture medium used in the following examples is a culture solution of the above-obtained preserved strain (Dehalobacter sp.) cultured at 30°C to the logarithmic growth phase in anaerobic inorganic salt liquid medium.

[0085] (1) Degradation curve of dichloromethane by dehalogenated bacteria strain DCM

[0086] The treatment group was set up as follows: 1 mL of the culture medium of the dehalogenated Bacillus strain DCM obtained above was inoculated into an anaerobic bottle containing 100 mL of anaerobic inorganic salt liquid medium, and 0.1 mL of compound vitamin mixture and 10 μL of dichloromethane were added to the culture bottle. The blank control group consisted of the same culture components except for the absence of the dehalogenated Bacillus strain DCM. All bottles in the treatment group and the blank control group were placed in a 30°C incubator for incubation. Periodically, 2 mL samples of bacterial culture were taken for analysis of the substrate and degradation products. One mL of each sample was used to quantitatively analyze the consumption of dichloromethane by the strain using gas chromatography, following the method in Case Study 1. The remaining 1 mL of each sample was centrifuged at 12000 × g at room temperature for 10 minutes. The supernatant was transferred to a 2 mL HPLC vial, acidified with 1 μL of concentrated sulfuric acid, and then placed in the HPLC sample tray to analyze the type and content of dichloromethane degradation products. The instrument parameters were as follows: Agilent 1260LC series HPLC; diode array detector (DAD), wavelength set to 210 nm; elution column: Aminex HPX-87H (300 × 7.8 mm, 9 μm, Bio-Rad, USA); flow rate: 0.6 mL / min; mobile phase: 100% 4 mM H₂SO₄; total run time: 20 min.

[0087] After a week of cultivation and monitoring, it was observed that the added dichloromethane content in the blank control group showed almost no decrease, and no products appeared, indicating that dichloromethane pollutants do not undergo a non-biodegradation reaction. Furthermore, the dichloromethane in the treatment group inoculated with the dehalogenated Bacillus strain DCM was rapidly consumed, with a degradation rate as high as 32 μmol / L. -1 Small molecule organic acid products with no environmental side effects were also detected in the bottle, namely 37.3 μmol acetic acid and 8.9 μmol formic acid. Figure 4 ).

[0088] (2) Maximum substrate amount for dichloromethane degradation by Dehalogenated Bacillus strain DCM

[0089] Different concentrations of dichloromethane (10, 20, 30, 40, 50, 100 μL) were added to anaerobic culture flasks containing 100 mL of anaerobic inorganic salt liquid medium using a microsyringe. The flasks were then sonicated at 100 W for 20 minutes in an ultrasonic cleaner to promote dichloromethane dissolution. Next, 0.1 mL of multivitamin complex (Table 1) and 1 mL of the obtained dehalogenated Bacillus strain DCM culture medium were added to all flasks as inoculum. All flasks were incubated at 30°C. Periodically, 1 mL samples of the bacterial culture were collected and analyzed by gas chromatography to monitor dichloromethane degradation.

[0090] After 20 days of cultivation and monitoring, it was observed that the *Dehalogenated Bacterium* strain DCM in the group with ≥30 μL of dichloromethane addition could completely degrade dichloromethane; while the *Dehalogenated Bacterium* strain DCM in the groups with addition amounts of 40 and 50 μL could only degrade 30% and 10% of the initial addition amount, respectively; dichloromethane in the group with 100 μL addition was not degraded. Therefore, in a 100 mL culture system, *Dehalogenated Bacterium* strain DCM can effectively degrade dichloromethane up to 30 μL, but dichloromethane exceeding 30 μL inhibits the growth of *Dehalogenated Bacterium* strain DCM.

[0091] (3) The optimal temperature for the degradation of dichloromethane by the dehalogenated bacillus strain DCM.

[0092] Take 1 mL of the above-obtained dehalogenated Bacillus strain DCM culture medium and inoculate it into a culture flask containing 100 mL of anaerobic inorganic salt liquid medium at a ratio of 1% (v / v). Add 10 μL of dichloromethane and compound vitamins, and then incubate at constant temperature and in the dark in incubators at 4℃, 10℃, 16℃, 20℃, 25℃, 30℃, 34℃, 37℃ and 40℃ respectively. The consumption of dichloromethane is monitored periodically by gas chromatography.

[0093] The results showed that after 20 days of culture, the *Dehalogenated Bacillus* strain DCM showed no dichloromethane degradation activity below 10℃; dichloromethane degradation was observed in the culture temperature range of 16-40℃; and the highest dichloromethane degradation rate was observed in the culture temperature range of 30-37℃. Figure 5 ).

[0094] Example 4. Degradation of environmental samples containing dichloromethane by the dehalogenated bacillus strain DCM

[0095] A 15L sample of laboratory waste water containing approximately 0.1mM dichloromethane was collected in a large plastic container (latitude: 41°54′28″, longitude: 123°35′42″). Sodium sulfide with a final concentration of 0.05mM was added to the water sample to remove oxygen. 1mL of the compound vitamins listed in Table 1 was also added. The culture medium of the dehalogenated Bacillus strain DCM obtained in Example 3 was centrifuged at 12000×g for 10 minutes to prepare a concentrated cell culture of the dehalogenated Bacillus strain DCM. This concentrated culture was then inoculated into the container containing the water sample at a ratio of 0.1% (w / v). The container was sealed with a rubber stopper and placed at 30°C in the dark for static incubation. During incubation, the consumption of dichloromethane in the system was monitored periodically using a gas chromatograph.

[0096] After half a month of cultivation, it was observed that dichloromethane in the laboratory waste water sample was completely consumed. This demonstrates that the degradation rate of dichloromethane in environmental samples using the dehalogenated Bacillus strain DCM of this invention can reach 100%, exhibiting excellent practical application results.

Claims

1. A dehalogenated bacillus strain capable of anaerobic degradation of dichloromethane, characterized in that: The strain is dehalogenated bacillus ( Dehalobacter (sp.)DCM was deposited on November 30, 2021, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 24007.

2. The dehalogenating bacteria with dichloromethane anaerobic degradation ability according to claim 1, characterized in that: The bacteria are rod-shaped, Gram-positive, 1-2 μm in length, and 0.3-0.5 μm in diameter.

3. An application of the dehalogenated Bacillus as described in claim 1, characterized in that: Application of the strain in degrading dichloromethane pollutants in the environment.

4. The application of the dehalogenated Bacillus according to claim 3, characterized in that: The environment is water, sediment, or soil.

5. An application of the dehalogenated Bacillus as described in claim 1, characterized in that: The application of the strain in degrading dichloromethane pollutants in the environment under anaerobic conditions.

6. A formulation for degrading dichloromethane pollutants, characterized in that: The formulation contains the dehalogenated Bacillus strain DCM as described in claim 1.

7. The formulation for degrading dichloromethane pollutants according to claim 6, characterized in that: The preparation contains cell cultures, cell culture concentrates, or culture suspensions of the strain.

8. A method for degrading dichloromethane pollutants, characterized in that: The strain described in claim 1 or the preparation described in claim 6 is inoculated into the environmental sample to be treated, and sodium sulfide at a final concentration of 0.05 mM is added to the environmental sample as an oxygen scavenger, thereby achieving the anaerobic degradation of dichloromethane in the environmental sample.

9. The method according to claim 8, characterized in that: The inoculation amount of the strain or preparation is 0.1% (w / v).

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