A method for remediation of halohydrocarbon pollution based on functional plasmid transformation
By transforming functional plasmid DNA into indigenous microorganisms, a bio-enhanced remediation system was constructed, solving the problems of slow degradation and ecological risks of halogenated hydrocarbon pollutants in existing technologies, and achieving efficient and safe pollutant degradation.
Patent Information
- Application Number
- CN202610166763.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-05
- Publication Date
- 2026-05-26
AI Technical Summary
Existing bioremediation technologies are slow to start, difficult to enrich functional bacterial strains, and difficult to colonize exogenous bacterial agents, making it difficult to completely degrade halogenated hydrocarbon pollutants and posing ecological risks.
Functional plasmid DNA was extracted from organic halogen-reducing bacteria, transformed into indigenous microorganisms, and a bio-enhanced remediation system was constructed to activate its reductive dechlorination function. Pollution remediation was carried out under anaerobic conditions using electron donors such as lactic acid.
It significantly accelerates the complete degradation of halogenated hydrocarbon pollutants, avoids the accumulation of toxic intermediate products, combines high efficiency with environmental safety, and avoids the ecological risks of introducing exogenous bacteria.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of environmental microbial remediation technology, specifically relating to a method for remediating halogenated hydrocarbon pollution based on functional plasmid transformation. Background Technology
[0002] Organochlorinated hydrocarbons (such as trichloroethylene, TCE) are common pollutants in soil and groundwater, characterized by high toxicity, strong mobility, and recalcitrant degradation. Under natural conditions, they can undergo reductive dechlorination reactions to generate even more toxic intermediates, such as cis / trans-1,2-dichloroethylene (cis / trans-DCE) and vinyl chloride (VC), posing a serious threat to the ecological environment and human health.
[0003] Currently, the remediation of contaminated sites mainly relies on physicochemical methods (such as adsorption and thermal desorption) and bioremediation methods. Physicochemical methods have problems such as high cost and high risk of secondary pollution; while bioremediation methods, especially natural attenuation technology based on microbial reduction and dechlorination, are widely used in Europe and the United States due to their advantages such as environmental friendliness, low cost, and in-situ implementation. Natural attenuation technology relies on organic halide respiring bacteria (OHRB) in the indigenous microbial community, such as obligate OHRB and facultative OHRB, to gradually reduce and dechlorinate high-chlorinated hydrocarbons through metabolism, ultimately converting them into harmless ethylene.
[0004] However, natural degradation technologies typically have long start-up periods, and in actual contaminated sites, the lack of key functional strains or insufficient microbial abundance often causes the dechlorination process to stall at the intermediate product stage, making complete degradation difficult. Furthermore, directly adding exogenous microbial agents may pose ecological risks, including low survival rates of inoculated strains and competitive disadvantages. Therefore, there is an urgent need to develop a bioaugmentation technology that can efficiently activate indigenous microbial communities, accelerate the dechlorination process, and control environmental risks.
[0005] In recent years, genetic biological enhancement has attracted attention as an emerging strategy. This technology introduces plasmids carrying key functional genes into indigenous microorganisms, enabling them to acquire reductive dechlorination capabilities, thereby improving repair efficiency and showing promising application prospects. Summary of the Invention
[0006] The purpose of this invention is to solve the problems of slow start-up, difficulty in enriching functional strains, and difficulty in colonizing exogenous bacterial agents in existing bioremediation technologies, and to provide a method for remediation of halogenated hydrocarbon pollution based on functional plasmid transformation.
[0007] The specific technical solution adopted in this invention is as follows:
[0008] In a first aspect, the present invention provides a method for remediating halohydrocarbon pollution based on functional plasmid transformation, as follows:
[0009] S1: Extracting functional plasmid DNA with reductive dechlorination function from organohalogen respiring bacteria; organohalogen respiring bacteria are microorganisms that use organohalogen compounds as electron acceptors for reductive dechlorination metabolism;
[0010] S2: Collect samples containing halogenated hydrocarbon pollutants and retain indigenous microorganisms in the samples;
[0011] S3: Add the functional plasmid DNA and electron donor to the sample to be treated to construct a bio-enhanced remediation system; place the bio-enhanced remediation system under anaerobic conditions for pollution remediation, and enhance its reductive dechlorination effect on halogenated hydrocarbon pollutants by transforming the functional plasmid DNA into indigenous microorganisms.
[0012] Preferably, the organic halogen respiration reducing bacteria are one or more of the following: Desulfitobacterium, Dehalococcoides, Dehalobacter, Geobacter, or Anaeromyxobacter.
[0013] Preferably, the organohalogen respiration reducing bacteria is Desulfitobacterium sp. DL, with accession number CCTCC M 20252484, accession date November 10, 2025, and deposited at the China Center for Type Culture Collection.
[0014] As a preferred embodiment, the functional plasmid extraction method in step S1 is as follows:
[0015] The organohalogenated respiratory bacteria were resuspended in a magnesium-containing buffer solution, and deoxyribonuclease I was added to degrade the extracellular free DNA. After degradation, EDTA was added to a final concentration of 5 mM, and the mixture was heated to 65°C to completely inactivate deoxyribonuclease I. Subsequently, total intracellular plasmid DNA of the organohalogenated respiratory bacteria was extracted using a plasmid extraction kit.
[0016] Furthermore, the plasmid extraction kit uses the Plasmid Midi Kit.
[0017] Preferably, the sample to be treated is soil, groundwater, or sediment containing organochlorine hydrocarbon pollutants.
[0018] Furthermore, the organochlorinated hydrocarbon pollutant is one or more of trichloroethylene, tetrachloroethylene, 1,1-dichloroethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, or vinyl chloride.
[0019] Preferably, the electron donor is one or more of lactic acid, sodium lactate, pyruvic acid, hydrogen, or formic acid.
[0020] Preferably, the volume of functional plasmid DNA added to the bio-enhanced repair system is 1% to 10% of the sample to be treated.
[0021] Preferably, in step S3, the oxidation-reduction potential of the pollution remediation process is controlled to be below -300 mV, and the temperature is controlled to be 25~35℃.
[0022] Compared with the prior art, the present invention has the following advantages:
[0023] This invention provides a functional plasmid transformation remediation technology. Functional plasmid DNA is extracted from organohalogen-reducing bacteria and then introduced into indigenous microorganisms in the soil to be treated, effectively activating the reductive dechlorination function of the indigenous microorganisms. The method provided by this invention significantly accelerates the complete degradation of halogenated hydrocarbon pollutants such as trichloroethylene (TCE), avoiding the accumulation of toxic intermediate products. This method combines high efficiency with environmental safety, avoiding the ecological risks of introducing exogenous bacteria, and is a novel in-situ bioremediation method with broad application prospects. Attached Figure Description
[0024] Figure 1 This is a graph showing the change in trichloroethylene content during the culture process after inoculation with organic halogenated respiratory reducing bacteria in Example 1;
[0025] Figure 2 The graph shows the changes in the contents of dichloroethylene, vinyl chloride, and ethylene during the culture process after inoculation with organic halogen-reducing respiratory bacteria in Example 1.
[0026] Figure 3 This is a comparison chart of the degradation effects of trichloroethylene (TCE) in different treatment groups in Example 2;
[0027] Figure 4 This is a comparison chart of the degradation effects of trichloroethylene (TCE) in different treatment groups in Example 3;
[0028] Figure 5 This is a comparison chart of the average dechlorination rate (a) and trichloroethylene removal rate (b) of different treatment groups in Example 2;
[0029] Figure 6 This is a comparison chart of the average dechlorination rate (a) and trichloroethylene removal rate (b) of different treatment groups in Example 3. Detailed Implementation
[0030] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.
[0031] The organohalogenated respiratory reducing bacteria used in the following examples is Desulfitobacterium sp. DL, with accession number CCTCC M 20252484, accession date November 10, 2025, and deposited at the China Center for Type Culture Collection.
[0032] Example 1
[0033] This embodiment provides a method for extracting functional plasmid DNA with reductive dechlorination function, as detailed below:
[0034] (1) Preparation of anaerobic enrichment culture medium
[0035] The inorganic salt mother liquor contains, per liter: 1.0 g NaCl, 0.2 g KH2PO4, 0.3 g NH4Cl, 0.3 g KCl, 0.015 g CaCl2·2H2O, and 0.06 g MgCl2·6H2O.
[0036] The trace element A solution contains, per liter: 0.006 g Na2SeO3·5H2O, 0.008 g Na2WO4·2H2O and 0.5 g NaOH.
[0037] The trace element B solution contains, per liter: 1.500 g FeCl2·4H2O, 0.190 g CoCl2·6H2O, 0.100 g MnCl2·4H2O, 0.070 g ZnCl2, 0.006 g H3BO3, 0.036 g Na2MoO4·2H2O, 0.024 g NiCl2·6H2O, 0.002 g CuCl2·2H2O, and 10 mL of 25% HCl solution.
[0038] Take 10 mL of the above inorganic salt mother liquor as the base solution, add 1 mL of trace element A solution and 1 mL of trace element B solution, then add 2.383 g (final concentration 10 mM) of HEPES organic buffer and 1.050 g (final concentration 10 mM) of NaHCO3 to adjust the pH to 7.2~7.5 to obtain the anaerobic enrichment medium.
[0039] (2) Deoxygenation and repackaging
[0040] After preparation, the anaerobic enrichment medium was boiled for 20 min under nitrogen protection to remove dissolved oxygen. It was then dispensed into 250 mL serum bottles, each containing 135 mL of the anaerobic enrichment medium. Each bottle was sealed with a PTFE-coated butyl rubber stopper and autoclaved at 121 °C for 30 min. After sterilization, the bottles were transferred to an anaerobic workstation for cooling and equilibration for 24 h to ensure a strictly anaerobic environment.
[0041] The headspace gas in the serum bottle was replaced three times with high-purity nitrogen (purity >99.99%) in the anaerobic workstation, and 1 mL of reducing agent solution (containing 0.024 g Na2S·9H2O, 0.048 g L-cysteine hydrochloride and 0.25 mL 0.1% v / v resazurin indicator solution per liter) was injected to completely remove residual oxygen. The solution was allowed to stand until the resazurin indicator solution turned colorless (redox potential Eh < -300 mV) and then the residual reducing agent was extracted.
[0042] Inject 0.3 mL of sterile multivitamin solution (ATCC MD-VS, USA) and 0.5 mg / L vitamin B12 solution into each serum bottle. Use a differential pressure gauge and an airtight syringe to precisely adjust the headspace pressure inside the bottle to 101.3 ± 0.1 kPa (standard atmosphere).
[0043] (3) Isolation of organic halogen-reducing bacteria
[0044] Each serum vial was supplemented with 250±50 µM trichloroethylene (TCE) as an electron acceptor and 10 mM sodium lactate as an electron donor and carbon source, and each vial was inoculated with 10% (v / v) anaerobic culture. The anaerobic culture was an enriched culture from sediments in the Three Gorges Reservoir area and sites contaminated with halogenated hydrocarbons.
[0045] After inoculation, the serum bottles were placed in the dark at 30°C for static incubation. During the incubation period, headspace gas was collected every 2-3 days using a 100 μL airtight syringe (Gaoge Industry & Trade Co., Ltd., Shanghai) to detect the concentrations of various chlorinated olefins and ethylene. When the vinyl chloride (VC) concentration fell below the detection limit (0.5 μM), trichloroethylene (TCE) was added to bring the initial concentration back up. After approximately 30 days of incubation, a bacterial culture with stable reductive dechlorination performance (average dechlorination rate ≥100 μM Cl) was obtained. - / d). Verification confirmed that the bacterial culture contained *Desulfitobacterium* sp. DL, with accession number CCTCC M 20252484.
[0046] The reducing and dechlorinating efficiency of this bacterial solution is as follows: Figure 1 and Figure 2 As shown, this demonstrates that the bacterium can be used as a plasmid extraction source and positive control inoculum for subsequent functional plasmid enhancement experiments.
[0047] (4) Plasmid extraction
[0048] Take 60 mL of organic halogen respiratory reducing bacterial culture, resuspend it in a magnesium-containing buffer solution, add deoxyribonuclease I (DNase I), and incubate at 37°C for 30-60 minutes to completely degrade extracellular free DNA. The purpose is to strictly eliminate extracellular DNA interference and accurately determine whether exogenous plasmids have entered the indigenous bacterial cells.
[0049] After degradation, EDTA was added to a final concentration of 5 mM, and the sample was heated at 65°C for 10 minutes to completely inactivate deoxyribonuclease I. Subsequently, the sample was washed and centrifuged multiple times with phosphate-buffered saline (PBS) to thoroughly remove the enzyme digestion products and inactivated enzyme protein.
[0050] Finally, the Plasmid Midi Kit (QIAGEN, USA) was used to extract plasmid DNA from the cells of the organohalogen respiring bacteria, following the manufacturer's instructions.
[0051] Example 2
[0052] This embodiment provides a method for remediating halogenated hydrocarbon pollution in pesticide-contaminated soil samples using plasmid DNA extracted in Example 1, as detailed below:
[0053] (1) Sample collection
[0054] Shallow soil samples (labeled WS) from pesticide-contaminated soil in Wuhan were collected. After pretreatment, the samples were stored at -20℃ to preserve the indigenous microorganisms in the soil samples.
[0055] (2) Repair the experimental setup
[0056] Control group (labeled WS-O group): A bio-enhanced remediation system was constructed in 250 mL serum bottles. 150 mL of anaerobic enrichment medium (prepared as in Example 1) was added to each bottle. 10 mM sodium lactate was used as an electron donor, and 10 g of contaminated soil sample was used as inoculum. The final concentration of trichloroethylene in the system was 300 μM.
[0057] Plasmid enhancement experimental group (labeled WS-E group): Prepare a plasmid stock solution with a concentration of 100 ng / μL, and add 5 μL of plasmid DNA liquid to the control group.
[0058] Each group had 3 biological replicates. All serum bottles were tightly sealed with butyl rubber stoppers and aluminum caps and incubated statically in a constant temperature and dark environment at 30°C for a total period of 91 days.
[0059] (3) Monitoring of indicators during the cultivation process
[0060] Headspace gas was periodically collected using a gas-tight syringe, and the concentration changes of TCE, its dechlorination intermediates, and the final product ethylene were analyzed by gas chromatography-FID to assess the reductive dechlorination kinetics of each system. Results are as follows: Figure 3 As shown.
[0061] according to Figure 3 It can be seen that the trichloroethylene content in the plasmid-enhanced experimental group with added plasmid DNA decreased significantly. The trichloroethylene removal rate is as follows: Figure 5 As shown in (b), the removal rate of trichloroethylene in the plasmid-enhanced experimental group was significantly higher than that in the control group.
[0062] A dechlorination kinetic model was constructed using MATLAB, and the experimental data were fitted to calculate the average dechlorination reaction rate. The results are as follows: Figure 5 As shown in (a), the dechlorination rate of the plasmid-enhanced experimental group was significantly improved, and the product conversion rate was close to complete degradation.
[0063] (4) Molecular biological analysis
[0064] At the end of the experiment, biological samples were collected, and total DNA and RNA were extracted. The abundance of functional genes (pceA, tceA, vcrA, etc.) and the evolution of microbial community structure were detected by qPCR, 16S rRNA sequencing and metagenomic analysis.
[0065] Example 3
[0066] This embodiment provides a method for remediating halogenated hydrocarbon pollution in ethylene-contaminated soil samples using plasmid DNA extracted in Example 1, as detailed below:
[0067] (1) Sample collection
[0068] Shallow soil samples (labeled HS) contaminated with chloroethylene were collected from a chlor-alkali plant in Hefei. After pretreatment, the samples were stored at -20℃ to preserve the indigenous microorganisms in the soil samples.
[0069] (2) Repair the experimental setup
[0070] Control group (labeled HS-O group): A bio-enhanced remediation system was constructed in 250 mL serum bottles. Each bottle contained 150 mL of anaerobic enrichment medium (prepared as in Example 1), 10 mM sodium lactate as an electron donor, and 10 g of contaminated soil sample as inoculum. The final concentration of trichloroethylene in the system was 280 μM.
[0071] Plasmid enhancement experimental group (labeled HS-E group): 5 μL of plasmid DNA liquid was added to the control group.
[0072] Each group had 3 biological replicates. All serum bottles were tightly sealed with butyl rubber stoppers and aluminum caps and incubated statically in a constant temperature and dark environment at 30°C for a total period of 91 days.
[0073] (3) Monitoring of indicators during the cultivation process
[0074] The collection methods and indicators are the same as in Example 2. The changes in trichloroethylene concentration in different treatment groups are as follows: Figure 4 As shown.
[0075] according to Figure 4 It can be seen that the trichloroethylene content in the plasmid-enhanced experimental group with added plasmid DNA decreased significantly. The trichloroethylene removal rate is as follows: Figure 6 As shown in (b), the removal rate of trichloroethylene in the plasmid-enhanced experimental group was significantly higher than that in the control group.
[0076] A dechlorination kinetic model was constructed using MATLAB, and the experimental data were fitted to calculate the average dechlorination reaction rate. The results are as follows: Figure 6 As shown in (a), the dechlorination rate of the plasmid-enhanced experimental group was significantly improved, and the product conversion rate was close to complete degradation.
[0077] (4) Molecular biological analysis, same as in Example 2.
[0078] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.
Claims
1. A method for remediating halohydrocarbon pollution based on functional plasmid transformation, characterized in that, Specifically as follows: S1: Extracting functional plasmid DNA with reductive dechlorination function from organohalogen respiring bacteria; organohalogen respiring bacteria are microorganisms that use organohalogen compounds as electron acceptors for reductive dechlorination metabolism; S2: Collect samples containing halogenated hydrocarbon pollutants and retain indigenous microorganisms in the samples; S3: Add the functional plasmid DNA and electron donor to the sample to be treated to construct a bio-enhanced remediation system; place the bio-enhanced remediation system under anaerobic conditions for pollution remediation, and enhance its reductive dechlorination effect on halogenated hydrocarbon pollutants by transforming the functional plasmid DNA into indigenous microorganisms.
2. The method for remediation of halogenated hydrocarbon pollution based on functional plasmid transformation according to claim 1, characterized in that, The organic halogen respiratory reducing bacteria are one or more of the following: Desulfitobacterium, Dehalococcoides, Dehalobacter, Geobacter, or Anaeromyxobacter.
3. The method for remediation of halogenated hydrocarbon pollution based on functional plasmid transformation according to claim 1, characterized in that, The organic halogen respiration reducing bacteria are desulfurobacillus (Bacillus thiocyanate). Desulfitobacterium sp. DL, accession number CCTCC M20252484, accession date November 10, 2025, is deposited at the China Center for Type Culture Collection.
4. The method for remediation of halohydrocarbon pollution based on functional plasmid transformation according to claim 1, characterized in that, The method for extracting functional plasmids in step S1 is as follows: The organohalogenated respiratory bacteria were resuspended in a magnesium-containing buffer solution, and deoxyribonuclease I was added to degrade the extracellular free DNA. After degradation, EDTA was added to a final concentration of 5 mM, and the mixture was heated to 65°C to completely inactivate deoxyribonuclease I. Subsequently, total intracellular plasmid DNA of the organohalogenated respiratory bacteria was extracted using a plasmid extraction kit.
5. The method for remediation of halohydrocarbon pollution based on functional plasmid transformation according to claim 4, characterized in that, The plasmid extraction kit used was the Plasmid Midi Kit.
6. The method for remediation of halohydrocarbon pollution based on functional plasmid transformation according to claim 1, characterized in that, The samples to be treated are soil, groundwater, or sediments containing organochlorine hydrocarbon pollutants.
7. The method for remediation of halogenated hydrocarbon pollution based on functional plasmid transformation according to claim 6, characterized in that, The organochlorinated hydrocarbon pollutant is one or more of trichloroethylene, tetrachloroethylene, 1,1-dichloroethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, or vinyl chloride.
8. The method for remediation of halogenated hydrocarbon pollution based on functional plasmid transformation according to claim 1, characterized in that, The electron donor is one or more of lactic acid, sodium lactate, pyruvic acid, hydrogen, or formic acid.
9. The method for remediation of halogenated hydrocarbon pollution based on functional plasmid transformation according to claim 1, characterized in that, The volumetric amount of functional plasmid DNA added in the bio-enhanced repair system is 1% to 10% of the sample to be treated.
10. The method for remediation of halohydrocarbon pollution based on functional plasmid transformation according to claim 1, characterized in that, In step S3, the redox potential of the pollution remediation process is controlled below -300 mV, and the temperature is controlled at 25~35℃.