A strain of dehalogenated monoclonal bacteria and its application in bioremediation of high-salinity environments

Abstract

This invention relates to the field of environmental microbial remediation, specifically the isolation and purification of a novel organohalogen-respirating dehalogenating bacterium (Dehalogenimonas) and its application in the bioremediation of chloroalkane contaminant 1,2-dichloroethane under high salinity conditions. The strain, *Dehalogenimonas* sp., was deposited on November 22, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40791. The degradation capacity of this strain under high salinity conditions not only allows for its application in the remediation of sites contaminated with chloroalkanes, particularly those with salinity gradients such as coastal, estuarine, and even marine environments, but also contributes to understanding the effects of salinity on the microbial reductive dehalogenation process and the molecular mechanisms of microbial tolerance to high salt. This research contributes to the development of innovative bioremediation technologies to address a range of halogenated contaminant pollution problems in saline-alkali environments.

Description

Technical Field

[0001] This invention relates to the field of environmental microbial remediation, specifically to the isolation and purification of a novel dehalogenated organic halogenated respiratory bacterium (Dehalogenimonas) and its application in the bioremediation of chloroalkane pollutant 1,2-dichloroethane under high salinity conditions. Background Technology

[0002] Chlorinated alkanes are an important class of chlorinated organic solvents, widely used as cleaning solvents for electronic components, intermediates in industrial chemical production, degreasing agents, and agricultural pesticides, exhibiting persistence in the environment. Among them, 1,2-dichloroethane is one of the most commonly used and frequently detected chlorinated alkanes. 1,2-Dichloroethane is a colorless, transparent, oily liquid with high lipid solubility, low flammability, high vapor pressure, and low boiling point. Industrially, it is often used as an industrial raw material, organic solvent, and petroleum additive in the chemical synthesis of polyvinyl chloride and other chemicals. Due to large-scale production and use, as well as illegal releases, accidental spills, equipment leaks, and improper disposal, 1,2-dichloroethane is widely present in groundwater and soil, and levels are severely exceeded. This not only poses a significant challenge to environmental remediation efforts but also poses a serious threat to human health due to its carcinogenic, teratogenic, and mutagenic effects, and has been listed as a "priority pollutant" in my country's environmental protection list.

[0003] Organohalide-respiring bacteria are the main force in the anaerobic degradation and bioremediation of organohalide pollutants in contaminated soil and groundwater. These bacteria share the characteristic of utilizing organohalides as electron acceptors and hydrogen or formic acid as electron donors, conducting reductive dehalogenation reactions (hydrogen atoms replacing halogen groups) through electron transfer to obtain the energy required for growth. This process is called "organohalide respiration" (OHR), and the dehalogenating bacteria involved are called organohalide-respiring bacteria (OHRB). The anaerobic degradation of 1,2-dichloroethane mainly occurs through a dihalogenation elimination process catalyzed by organohalide-respiring bacteria, removing two adjacent chlorine atoms to form a carbon-carbon double bond, with the final product being non-toxic ethylene. Currently, several organohalide-respiring bacteria capable of anaerobic degradation of 1,2-dichloroethane have been isolated from contaminated sites, such as *Dehalococcoides mccartyi* strain 195. T And BAV1, Desulfitobacterium dichloroeliminans strain DCA1 TAnd *Desulfitobacterium* strain AusDCA, *Dehalobacter* sp. strain WL, *Geobacter* strain IAE, and *Dehalogenimonas lykanthroporepellens* strains BL-DC-8 and BL-DC-9 T ,(Dehalogenimonas alkenigignens) strain IP3-3 T and SBP-1, (Dehalogenimonas formicexedens) strain NSZ-14 T The strain *Dehalogenimonas formicexedens* (GP) was also mentioned. However, all of the above strains were isolated from terrestrial environments, and there are no reports on the existence of organohalogen-respiring bacteria capable of growing and utilizing 1,2-dichloroethane in marine or estuarine environments.

[0004] Organohalogenating bacteria-mediated in-situ bioremediation has been successfully applied to clean up non-saline sites contaminated with halogenated hydrocarbons (HHH), typically with low salinity (<0.5 g / L). However, halogenated hydrocarbon pollution is not limited to terrestrial environments. Increasing population pressure, urbanization along the coast, and nutrient and sediment runoff from agricultural and forestry areas are significantly impacting the ecological changes of coastal and estuarine systems, leading to pollution and eutrophication in marine and estuarine environments. Naturally generated or anthropogenically released HHH in the marine environment can serve as growth substrates for certain microbial populations (e.g., organohalogenating bacteria), but their biodiversity and ecological functions remain to be further elucidated. Meanwhile, high-throughput sequencing results indicate that the genomes of various microorganisms from the marine environment encode reductive dehalogenase genes, but biochemical and physiological evidence proving that these microorganisms can perform organohalogen respiration remains limited. Furthermore, it has been reported that salinity is a key environmental factor influencing the structure, function, and composition of microbial communities in coastal wetlands, rivers, and saline-alkali soils with natural salinity gradients. However, organohalogen-respiring bacteria, such as *Dehalogenimonas lykanthroporepellens*, from both non-saline terrestrial and saline marine environments, exhibit severely inhibited dechlorination activity under environmentally relevant high salinity conditions (e.g., >20 g / L). Although several organohalogen-respiring bacteria capable of degrading 1,2-dichloroethane have been isolated, only the *Dehalogenimonas lykanthroporepellens* strain BL-DC-9 has shown significant dechlorination activity. T No organohalogenated bacteria that can grow in 2% sodium chloride solution concentrations or utilize 1,2-dichloroethane have been reported to grow at concentrations above 2% sodium chloride.

[0005] Therefore, obtaining microbial agents that can utilize chlorinated alkane pollutants in high-salinity environments (>2% sodium chloride salinity) such as oceans or estuaries, dechlorinate and transform them to generate non-toxic end products, is an important requirement for implementing in-situ environmental remediation projects. Summary of the Invention

[0006] The purpose of this invention is to provide the isolation and purification of a novel dehalogenated organic halogenated respiratory bacterium (Dehalogenimonas) and its application in the bioremediation of chloroalkane contaminant 1,2-dichloroethane under high salinity conditions.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A strain of dehalogenated monoclonal bacteria, namely Dehalogenated Monoclonal bacteria (Dehalogenated Monoclonal bacteria) (abbreviated as strain W), was deposited at the China General Microbiological Culture Collection Center on November 22, 2023, with the accession number CGMCC No. 40791.

[0009] The strain W was isolated from estuarine sediment samples at the mouth of the Wulihe River in Huludao, Liaoning Province, and was grown in an anaerobic inorganic salt medium containing 1,2-dichloroethane, acetic acid, and hydrogen.

[0010] The isolation and culture technique for strain W includes the following steps: enrichment and subculturing ten times in an anaerobic inorganic salt medium containing estuarine sediments and supplemented with 1,2-dichloroethane, acetic acid, and hydrogen. Using this mixed bacterial culture as the inoculum, an approximately pure culture is obtained using the extinction dilution method. This culture is then transferred to a medium supplemented with vancomycin (0.1 g / L) and ampicillin (1.0 g / L), maintaining growth conditions with 1,2-dichloroethane as the electron acceptor, and undergoes multiple subculturings.

[0011] An application of the aforementioned dehalogenated monoclonal bacterium, specifically the application of strain W in the degradation of chlorinated alkane contaminants.

[0012] Application of strain W in the degradation of chlorinated alkane pollutants under high salinity conditions.

[0013] The high salinity conditions refer to a 5% sodium chloride solution concentration.

[0014] The chlorinated alkane pollutant is 1,2-dichloroethane.

[0015] A bacterial agent for degrading 1,2-dichloroethane, the bacterial agent containing the aforementioned dehalogenated monoclonal bacteria (hereinafter referred to as strain W).

[0016] The bacterial agent is one or more of the following: culture, suspension, concentrate, and separation liquid of the bacterial strain.

[0017] The strain was cultured in an anaerobic inorganic salt medium with a carbon source, electron donor, and electron acceptor, and the organic halogenated dehalogenated monoclonal bacteria strain W was inoculated and cultured at pH 7.2, 30°C, and in the dark. The carbon source was acetic acid, the electron donor was hydrogen, and the electron acceptor was 1,2-dichloroethane.

[0018] The application of the aforementioned microbial agent for degrading 1,2-dichloroethane, specifically its application in the degradation of chloroalkane pollutants; or its application in the degradation of chloroalkane pollutants under high salinity conditions. Advantages of this invention:

[0019] The strain W obtained in this invention is a high-salt-tolerant 1,2-dichloroethane-degrading dehalogenated monoclonal bacterium screened from nearshore estuarine sediments.

[0020] Strain W is a Gram-negative bacterium. Through 16S rRNA gene sequence similarity comparison, it showed a species similarity of 97.5% with strain WBC-2 of the genus Dehalomonas, proving that strain W belongs to the genus Dehalomonas.

[0021] This strain can degrade 1,2-dichloroethane to ethylene under conditions of 30°C, pH 7.2, and 0.1-5% sodium chloride concentration. Its degradation capacity under high salinity conditions not only allows for the remediation of sites contaminated with chloroalkanes, particularly those with salinity gradients such as coastal, estuarine, and even marine environments, but also contributes to understanding the effects of salinity on microbial reductive dehalogenation processes and the molecular mechanisms of microbial tolerance to high salt. This research aids in the development of innovative bioremediation technologies to address a range of halogenated contaminants in saline-alkali environments. Attached image description:

[0022] Figure 1 Here is a scanning electron microscope image of strain W of the present invention;

[0023] Figure 2 A phylogenetic tree was constructed by homology comparison of the 16S rRNA gene sequences of strain W of the present invention and representative strains of the genus Dehalomonas.

[0024] Figure 3 The anaerobic degradation curve of 1,2-dichloroethane by strain W of the present invention is shown.

[0025] Figure 4 The anaerobic degradation curve of 1,2-dichloroethane by strain W of the present invention under 5% salinity conditions is shown. Detailed implementation method:

[0026] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0027] Unless otherwise specified, the experimental methods, materials and reagents used in the following examples are all conventional methods.

[0028] Example 1: Isolation, purification and identification of strains

[0029] (1) Prepare the basic culture medium:

[0030] Each liter of inorganic salt anaerobic culture medium contains: NaCl 1.0g, MgCl2·6H2O 0.5g, KH2PO4 0.2g, NH4Cl 0.3g, KCl 0.3g, CaCl2·2H2O 0.015g, FeCl2·4H2O 1.5mg, CoCl2·6H2O 190μg, MnCl2·4H2O 100μg, ZnCl2 70μg, H3BO3 6μg, Na2MoO4·2H2O 36μg, NiCl2·6H2O 24μg, CuCl2·2H2O 2μg, Na2SeO3·5H2O 6μg, Na2WO4·2H2O 8μg, and sodium resazurin 0.025% (w / v) as an oxygen indicator. The headspace of the culture medium was heated to boiling under high-purity N2 and then cooled to room temperature in an ice bath. 24 mg cysteine, 48 mg Na2S·9H2O, and 77 mg dithiothreitol (DTT) were added as reducing agents to remove oxygen. 2.52 g (30 mM) of NaHCO3 was added as a buffer. The pH of the medium was adjusted to 7.2-7.3 with CO2. The medium was then aliquoted into serum bottles and autoclaved at 121°C for 30 minutes. A multivitamin was then added. The final concentrations of each vitamin in the 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, and vitamin B12. 12 50 μg / L, p-aminobenzoic acid 50 μg / L, thioctic acid 50 μg / L.

[0031] (2) Enrichment by anaerobic bacteria that degrade 1,2-dichloroethane:

[0032] Dispense 80 mL of the anaerobic inorganic salt culture medium described in step (1) above into a 120 mL serum bottle, add 5 mM sodium acetate as a carbon source and 10 mL hydrogen as an electron donor, and add 6 μL (0.93 mM liquid concentration) of 1,2-dichloroethane as an electron acceptor through a microsyringe. In an anaerobic glove box, inoculate ~5 g of near-shore estuarine sediment (collected from Wulihe, Huludao City, Liaoning Province), and seal the serum bottle with a blue rubber stopper and aluminum cap to establish an enrichment culture system. Incubate at 30℃ in the dark, and monitor the degradation process of 1,2-dichloroethane periodically using gas chromatography. After 1,2-dichloroethane is completely degraded into ethylene, transfer to the same culture medium at a 3% (v / v) inoculation rate more than 5 times to simplify the community structure and highly enrich the anaerobic degrading bacteria of 1,2-dichloroethane.

[0033] (3) Isolation of anaerobic bacteria that degrade 1,2-dichloroethane:

[0034] Dispense 9 mL of the inorganic culture medium described in step (1) into a 20 mL culture flask, replace the headspace gas with N2 / CO2 (80 / 20, v / v), seal with a blue rubber stopper and aluminum cap, and add 2 μL of 1,2-dichloroethane using a microsyringe. Transfer 1 mL of the enriched culture of the 1,2-dichloroethane-degrading bacteria from the 120 mL serum bottle 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 bottle. Wait 10... -10 After the 1,2-dichloroethane in the diluted culture medium was completely degraded to ethylene, 1 mL was transferred to a medium supplemented with vancomycin (0.1 g / L) and ampicillin (1.0 g / L). The growth conditions, using 1,2-dichloroethane as the electron acceptor, were maintained. The above steps were then followed to establish a 10-year growth cycle. -1 The dilution was repeated multiple times in the dilution bottle, and the resulting dilution was inoculated into 80 mL of anaerobic inorganic salt medium containing 1,2-dichloroethane and incubated statically at 30°C in the dark. 1,2-dichloroethane-degrading bacteria were screened by monitoring the degradation of 1,2-dichloroethane.

[0035] (4) Identification of strains:

[0036] Morphological identification:

[0037] The obtained 1,2-dichloroethane bacterial culture was centrifuged at 4°C and 15000x g for 10 minutes to collect cells in the logarithmic growth phase. After fixing the cells with 2.5% glutaraldehyde and dehydrating them, the cell morphology was observed under an electron microscope (see [link to article]). Figure 1 The results showed that the strain W cells isolated by the above steps were irregularly round in planar appearance, with a diameter between 0.4 μm and 0.8 μm, and no flagella were observed.

[0038] 16S rRNA sequence homology analysis:

[0039] Total DNA was extracted from the strain and used as a template for amplification. PCR amplification was performed using universal primers 27F (5”-AGAGTTTGATCCTGGCTCAG-3”) and 1492R (5”-GGTTACCTTGT TACGACTT-3”) for bacterial 16S rRNA. The amplified products were submitted to a sequencing company for Sanger sequencing. The PCR-absorbed 16S rRNA gene fragment was 1377 bp, with a G+C mol% of 52.9%. Sequence alignment analysis showed that strain W had the highest sequence homology with the type strain BL-DC-9 of the genus *Lykanthroporepellens*, with a similarity of 97.5% (see [link to original text]). Figure 2 However, strain BL-DC-9 can only tolerate 2% salinity for growth, and its ability to tolerate higher salinity has not been reported. The 16S rRNA sequence of strain W is as follows:

[0040] CTTATGCATGCAGTCGACGGTCTCTCGCAAGAGAGATAGTGGCAAAC

[0041] GGGTGAGTAATAGATAAATAACCTGCCTTTAAGTGGGGGATAACACT

[0042] TCGAAAGAAGTGCTAATACCGCATGTGATGCTCTTTCATAAGAAGGA

[0043] TCATTAAAACCGCAAGGTGCTTGAAGAGGGGTTTGTCTCCGATTAGC

[0044] TTGTTGGTGGGGTAACGGCCTACCAAGGCAATGATCGGTAGCTGGTC

[0045] TGAGAGGATGGTCAGCCACACTGGAACTGAGACACGGTCCAGACTC

[0046] CTACGGGAGGCAGCAGCAAGGAATCTTGGGCAATGGGCGAAAGCCT

[0047] GACCCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAAACC

[0048] TCTTTTCTCAGGGAAGAATAATGACGGTACCTGAGGAATAAGTCTCG

[0049] GCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGAGGCGAGCGTT

[0050] ATCCGGATTTATTGGGCGTAAAGTGGGCGTAGGTGGTCTTTCAAGTC

[0051] AGATGTGAAATCTCCCGGCTCAACTGGGAGGGGTCATCTGATACTGT

[0052] TGGACTTGAGTATGGCAGGAGAAAACGGAATTCCCGGTGTAGTGGTG

[0053] AAATGCGTAGATATCGGGAGGAACACCAGAGGCGAAGGCGGTTTTC

[0054] TAGGCCAAAACTGACACTGAGGCCCGAAAGCGTGGGGAGCGAACAG

[0055] GATTAGATACCCTGGTAGTCCACGCCCTAAACACTGGGTACTAGGTA

[0056] TAGGGAGTATCGACCCTTTCTGTGCCGAAGCTAACGCTTTAAGTACC

[0057] CCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAAGGAATTGAC

[0058] GGGGGCCCGCACAAGCAGCGGAGCGTGTGGTTTAATTCGATGCTACA

[0059] CGAAGAACCTCACCAGGGTTTGACATGTTAGAAGTAGTGAACCGAA

[0060] AGGGGAACGACCTGTTAAGTCAGGAGCTATCACAGGTGCTGCATGGC

[0061] TGTCGTCAGCTCGTGCCGTGAGGTGTATGGTTAAGTCCTGCAACGAG

[0062] CGCAACCCTCATTGCTAGTTATATTCTCTAGCGATACTGCCTCGCAAA

[0063] ACGGGGAGGAAGGTGGGGATGACGTCAAGTCAGCATGGCCCTTATA

[0064] CCCTGGGCTACACACACGCTACAATGGGCGGTACAATGGGTTGCCAC

[0065] CGGGTGACCGGGAGCTAATCCCCAAAACCGCCCTCAGTTCGGATTGC

[0066] AGGCTGAAACTCGCCTGCATGAAGTCGGAGTTGCTAGTAAACGCGTG

[0067] TCAGCATAGCGCGTTGAATACGTTCTCGGGCCTTGTACACACCGCCC

[0068] GTCACGTCATGAAAGTTGGTAACACCTGAAGTCGATAGGCTAACCCG

[0069] CAAGGGAGGCAGTCGCCCAA

[0070] The strain is an organohalogenated dehalogenated monoclonal bacteria strain W, which was deposited on November 22, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40791, hereinafter referred to as strain W.

[0071] Example 2: Identification of the degradation performance of strain W on 1,2-dichloroethane

[0072] Add 5 mM sodium acetate as a carbon source and 10 mL hydrogen as an electron donor to the 80 mL anaerobic inorganic salt basal medium described in step (1) above. Add 6 μL (0.93 mM liquid phase concentration) of 1,2-dichloroethane as an electron acceptor through a microsyringe. Inoculate strain W at 3% (v / v) and incubate at pH 7.2 and 30°C in the dark.

[0073] 1,2-dichloroethane and its degradation products were determined using gas chromatography (Agilent 7890B) with a tandem flame ionization detector (FID) mounted on an Agilent DB-624 capillary column (60 m × 0.32 mm × 1.8 μm). The gas chromatographic parameters are as follows:

[0074] The injection port temperature was 200℃; the temperature program was: 60℃, hold for 2 min; then increase to 200℃ at a rate of 25℃ / min, hold for 1 min; the carrier gas was helium, and the column flow rate was 3 mL / min; the FID detector temperature was 300℃; the fuel gas was hydrogen, flow rate 30 mL / min; the combustion oxidizer was synthetic air, flow rate 350 mL / min; and the make-up gas was nitrogen, flow rate 25 mL / min. The autosampler parameters were as follows: GC cycle time 18 min, sample vial equilibration time 15 min, injection duration 0.5 min; oven temperature 70℃, quantitative loop temperature 125℃, and transfer line temperature 135℃.

[0075] Qualitative and quantitative analysis of the parent compound and degradation products was performed based on retention time and peak area. Figure 3 ).

[0076] Analysis showed that strain W could completely degrade 1,2-dichloroethane (liquid phase concentration 0.93mM) into non-toxic ethylene in a cycle of about 7 days, with an average degradation rate of 9.67 μmol / d.

[0077] Example 3: Degradation performance of strain W on 1,2-dichloroethane under 5% salinity conditions

[0078] The 80 mL anaerobic inorganic salt basal medium described in step (1) above was supplemented with 5 mM sodium acetate as a carbon source and 10 mL hydrogen gas as an electron donor. 6 μL (0.93 mM liquid concentration) of 1,2-dichloroethane was added via a microsyringe as an electron acceptor. An additional 5% sodium chloride solution was added, and strain W was inoculated at 3% (v / v). The culture was then incubated statically at pH 7.2 and 30°C in the dark. Quantitative monitoring of 1,2-dichloroethane and its degradation products was performed using the above gas chromatography conditions. Figure 4 ).

[0079] The analysis results showed that strain W could completely degrade 1,2-dichloroethane (liquid phase concentration 0.93mM) into non-toxic ethylene under 5% salinity conditions, with a degradation cycle of approximately 42 days and an average degradation rate of 1.61 μmol / d.

[0080] The bacterial agent was prepared according to the above description. For example, sodium acetate was added to an anaerobic inorganic salt culture medium as a carbon source, 10 mL of hydrogen gas as an electron donor, and 1,2-dichloroethane as an electron acceptor. The bacterial strain W was inoculated at 3% (v / v) and cultured at pH 7.2, 30°C, and in the dark for 5-7 days. The resulting culture was centrifuged, the precipitate was collected, and resuspended to obtain a resuspension. The liquid phase was the separation liquid. The culture was concentrated to obtain a concentrate, which is the bacterial agent. It has good application prospects for the bioremediation of groundwater contaminated with chloroalkanes or contaminated sites such as marine estuaries.

[0081] The above-described embodiments 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, characterized by: The dehalogenated monoclonal bacteria is strain W, which was deposited at the China General Microbiological Culture Collection Center on November 22, 2023, with the accession number CGMCC No. 40791.

2. The application of the dehalogenated monoclonal bacteria according to claim 1, characterized in that: Application of strain W in the degradation of chlorinated alkane pollutants; The chlorinated alkane pollutant is 1,2-dichloroethane.

3. The application of the dehalogenated monoclonal bacteria according to claim 2, characterized in that: Application of strain W in the degradation of chloroalkane pollutants under high salinity conditions; the high salinity conditions are 5% sodium chloride concentration; the chloroalkane pollutant is 1,2-dichloroethane.

4. A bacterial agent for degrading 1,2-dichloroethane, characterized in that: The microbial agent contains the dehalogenated monoclonal bacteria as described in claim 1.

5. The use of the bacterial agent according to claim 4 in the degradation of 1,2-dichloroethane.

6. The application of the bacterial agent of claim 4 in the degradation of 1,2-dichloroethane under high salinity conditions, wherein the high salinity conditions are a sodium chloride concentration of 5%.

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