Mixed bacterial agent for realizing efficient reductive dehalogenation and methane emission reduction
By constructing a microbial electron transport network in a mixed microbial agent, the problems of low dehalogenation efficiency and high methane emissions in contaminated sites were solved, achieving efficient pollution remediation and low carbon emissions.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QINGDAO UNIV
- Filing Date
- 2025-12-11
- Publication Date
- 2026-04-28
AI Technical Summary
Existing technologies are insufficient for efficient dehalogenation and reduction of methane emissions, resulting in low remediation efficiency and high carbon emissions for contaminated sites.
A mixed microbial agent containing *Dehalogenated Coccidia*, *Desulfovibrio*, *Clostridium*, *Peter's Cells*, *Methanogenic Bacteria*, and *Methane-oxidizing Bacteria* is used to improve the dehalogenation rate and reduce methane emissions by constructing an efficient electron transport network.
It significantly improved the dehalogenation rate, reduced methane emissions, and achieved green and low-carbon remediation of contaminated sites. The dehalogenation cycle of trichloroethylene was shortened from 10 days to 8 days, and methane emissions were reduced by 55.25%.
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Figure CN121930968A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioremediation technology for organic contaminated sites, and mainly relates to a mixed microbial agent that can improve the dehalogenation rate of halogenated organic matter and reduce methane emissions, thereby achieving green and low-carbon remediation of contaminated sites. Background Technology
[0002] Chlorinated olefins are widely used as solvents for dry cleaning and degreasing metal parts, but their entry into soil and groundwater environments can easily cause pollution. As heavy, non-aqueous liquids, they migrate downwards in soil and groundwater, causing continuous pollution to the soil and aquifers during their flow. Once pollution forms, it is difficult to completely dissolve and can only be released into the surrounding environment very slowly. For example, the half-life of trichloroethylene in soil is about six months to one and a half years, while its half-life in groundwater can be as long as eleven months to four and a half years. In addition, chlorinated olefins can also be transported through the groundwater cycle, thereby polluting other ecosystems.
[0003] In natural environments, the reductive dehalogenation of most halogenated organic pollutants is often accompanied by biological redox processes of some biogenic elements, such as methanogenesis, which is related to global carbon emissions. Both are important processes in anaerobic environments, affecting pollution reduction and global greenhouse gas emissions, respectively. From the perspective of elemental coupling processes, the reductive dehalogenation process of dehalogenating bacteria is generally positively correlated with methanogenesis, indicating a clear mutually beneficial relationship between the two processes. Methanogens can generally be divided into hydrogen-trophic, acetate-trophic, and methyl-trophic methanogens, which can create suitable growth conditions for dehalogenating *Streptococcus* to enhance reductive dehalogenation. From the perspective of electron donors, both hydrogen-trophic methanogens and dehalogenating *Streptococcus* can utilize hydrogen produced by the fermentation of substrates such as lactic acid by other fermenting bacteria (such as *Desulfovibrio*). For example, hydrogen-trophic methanogens can consume high concentrations of hydrogen (≤77 nM) and gradually reduce it to the suitable range for dehalogenating *Streptococcus* (≤2.5 nM), achieving efficient utilization of hydrogen in the reductive dehalogenation and methanogenesis processes, resulting in the production of large amounts of methane during reductive dehalogenation.
[0004] Anaerobic methane oxidation (AOM), as a "reverse methanogenesis" process in ecosystems, plays a crucial role in anaerobic ecosystems and is significant for balancing global methane concentrations and mitigating global warming. In soil systems with high methane emission fluxes, calculated based on air-dried soil, the flux of AOM converting methane to carbon dioxide reaches 0.15–1.30 µg C / g, with the specific conversion rate depending on the types of coexisting electrons (e.g., Fe). 3+ NO 3- SO4 2- It is estimated that global paddy field soils rely on NO. 3-The action of AOM (alternating current-oxidizing bacteria) consumes approximately 3900 Tg C–CH4 annually, offsetting about 10% to 20% of global methane emissions. Existing research on AOM as an electron-contributing process largely focuses on its close correlation with processes such as sulfate reduction and nitrate reduction; however, coupling AOM with reductive dehalogenation is more feasible. Firstly, in interspecies hydrogen transfer, hydrogen-trophic bacteria (such as sulfate-reducing bacteria and dehalogenating bacteria) consume hydrogen produced by methane oxidation to a lower partial pressure. At the corresponding in-situ concentrations of reactants and products, reductive dehalogenation can maintain a low theoretical H2 consumption threshold concentration of 7.19 × 10⁻⁶. -25 The nM value indicates that reductive dehalogenation and AOM processes can cooperate well. From a thermodynamic perspective, reductive dehalogenation with AOM produces a larger free energy change (ΔGº') than sulfate reduction. Under standard conditions, the ΔGº' of AOM is +135.4 kJ / mol CH4, while the ΔGº' of reductive dechlorination ranges from -127 to -193 kJ / mol H2, significantly larger than that of sulfate reduction (ΔGº' = -38 kJ / mol). These factors promote the interaction between the methane cycle and organohalogens in the anaerobic environment. Based on the electron transfer pathways between reductive dehalogenation, methanogenesis, and AOM, constructing an efficient microbial community system that facilitates the interaction of these three processes can provide technical support for the green and low-carbon remediation of sites contaminated by halogenated pollutants. Summary of the Invention
[0005] This invention provides a mixed microbial agent based on existing technology that can achieve efficient reductive dehalogenation and methane emission reduction, thereby improving the reductive dehalogenation rate of pollutants and reducing methane emissions. The mixed microbial agent comprises a microbial strain concentration of 9.68 × 10⁻⁶. 8 -2.82×10 9 dehalogenated Bacteroides (copy / mL) Dehalococcoides mccartyi ), 5.66×10 8 -6.21×10 8 desulfuric vibrio (copy / mL) Desulfovibrio desulfuricans ), 8.22×10 7 -1.25×10 8 Clostridium perfringens (copy / mL) Clostridium sp. ), 5.24×10 7 -3.43×10 8 Peterus spp. (copy / mL) Petrimonas mucosa ), 7.25×10 7 -1.38×10 8 Methanogens (copy / mL) Methanobacterium formicicum ) and 2.24×10 8 -4.29×10 8copy / mL of methanogenic bacteria ( Mtehylocystis bryophila This mixed microbial agent can be directly injected into sites contaminated with halogenated organic compounds. The various microorganisms in the microbial community construct an efficient electron transport network through complex nutrient interactions, enhancing microbial growth and metabolic activity, increasing the rate of dehalogenation by *Bacillus dehalogenatus*, and effectively reducing methane emissions from the system. When dehalogenation is complete, the dehalogenation rate can be increased by 20.00%, while methane production in the system can be reduced by up to 55.25%, which helps to achieve green and low-carbon remediation of sites contaminated with organic halogenated compounds.
[0006] The main principle of this invention is that, in real-world environments, reductive dehalogenation and methanogenesis are typically positively correlated; that is, dehalogenating bacteria and methanogenic bacteria have a positive interaction, resulting in efficient reductive dehalogenation while simultaneously producing large amounts of the greenhouse gas methane. Based on the functional interactions between microorganisms, methanogenic bacteria are introduced into the existing dehalogenation bacterial community. By consuming the methane concentration in the system, this further promotes the reductive dehalogenation reaction, achieving green and low-carbon reduction.
[0007] Based on the above principles, the inventor provides the following specific technical solution: A mixed microbial agent for achieving efficient reductive dehalogenation and methane emission reduction, in an environment with water as the solvent, comprises dehalogenated Bacillus, desulfurized Vibrio, Clostridium, Peterus mucosae, methanogens, and 2.24 × 10⁻⁶ ppm. 8 -4.29×10 8 Methanogenic bacteria (copy / mL).
[0008] Furthermore, the concentration of dehalogenated Bacteroides strain was 9.68 × 10⁻⁶. 8 -2.82×10 9 The concentration of *Vibrio desulfurans* was 5.66 × 10⁻⁶ copies / mL. 8 -6.21×10 8 The concentration of Clostridium difficile was 8.22 × 10⁻⁶ copies / mL. 7 -1.25×10 8 The concentration of Peterus mucosa was 5.24 × 10⁻⁶ copies / mL. 7 -3.43×10 8 The concentration of methanogenic bacteria was 7.25 × 10⁻⁶ copies / mL. 7 -1.38×10 8 copy / mL.
[0009] The above-mentioned bacterial agent can be either solid or liquid; when the bacterial agent is solid, the concentration of the above-mentioned bacterial strain is measured by dissolving it in water.
[0010] After research, the inventors discovered that, taking trichloroethylene as an example, compared to dehalogenation bacteria without methanogenic bacteria, the mixed bacterial community used in this invention can shorten the original dehalogenation cycle from 10 days to 8 days, and reduce the methane emission concentration from 388.88 µmol / L to 174.03 µmol / L after 8 days. While the methanogenic bacteria oxidize methane to reduce the methane concentration, they also produce intermediate products such as methanol and formic acid, which provide electron donors for the dehalogenating bacteria and can also perform cometolytic degradation of the toxic intermediates of trichloroethylene, thus accelerating the dechlorination of trichloroethylene.
[0011] The candidate strains in the above mixed bacterial agent are: *Dehalogenated Coccidia* (ATCCBAA-2266), *Desulfovibrio* (KCTC5768), *Clostridium* (ATCC33052), *Peter's bacillus* (CCAM323), *Methanogens* (ATCC33274), and *Methane-oxidizing bacteria* (NMDC20227199). The numbers of each strain are obtained from the National Microbial Science Data Center. Among them, *Peter's bacillus* and *Methane-oxidizing bacteria* can be purchased from Ningbo Mingzhou Biotechnology Co., Ltd., and the other strains can be purchased from Gray Algae Biotechnology Co., Ltd. All of them are commonly used strains in this field, and the inventors will not elaborate further.
[0012] In this invention, the pollutants in the organically contaminated site used for verification experiments are mainly chlorinated olefins. Taking trichloroethylene as an example, when the trichloroethylene content does not exceed 500 µmol / L and the chloride ion concentration corresponding to trichloroethylene does not exceed 1500 µmol / L, the total bacterial concentration in the treatment system is controlled to be 1.97 × 10⁻⁶ in this application. 8 -4.48×10 8 For other chlorinated alkenes, the bacterial concentration should be determined based on the chloride ion concentration, ensuring that the total bacterial concentration is excessive.
[0013] When applied to the remediation of actual contaminated sites, the dosage of the microbial agent can be adjusted appropriately according to the actual concentration of the pollutants. Generally, the concentration of chloride ions contained in the pollutants is used as the standard. As mentioned above, when the chloride ion concentration from chloroolefins does not exceed 1500 µmol / L, the corresponding total concentration of microbial community is controlled at 1.97 × 10⁻⁶ in this application. 8 -4.48×10 8 The concentration of the bacterial agent can be increased appropriately based on the actual site conditions to achieve an excess of bacteria. In addition to chlorinated olefins, the pollutants mentioned can also be other halogenated olefins or other halogen-containing pollutants. The dosage can be based on chlorinated olefins, and the concentration of other halogen elements can be used to replace the chloride ion concentration.
[0014] The aforementioned chlorinated olefins are selected from one or more of tetrachloroethylene, trichloroethylene, 1,1-dichloroethylene, trans-1,2-dichloroethylene, cis-1,2-dichloroethylene, and vinyl chloride. The mixed microbial agent targeted in this invention can treat systems where the concentration of chloride ions or other halide ions contained in halogenated olefins is below 1500 µmol / L. If the contamination concentration at individual sites exceeds this range, existing technologies such as gas-phase extraction can be used to reduce the concentration to this range before microbial community reduction and dehalogenation remediation.
[0015] In summary, compared to dehalogenation bacteria without methanogenic bacteria, the mixed bacterial agent used in this invention can enhance the rate of dehalogenation by *Bacillus dehalogenatus* and effectively reduce methane emissions. While dehalogenation bacteria without methanogenic bacteria require a 10-day degradation cycle to degrade TCE, the mixed bacterial agent provided in this application can completely convert TCE into the non-toxic product ethylene within as little as 8 days, and reduce methane production by up to 55.25%. Attached Figure Description
[0016] Figure 1 The reduction dehalogenation rate of the mixed bacterial community with different concentrations of methanogenic bacteria added in Example 3 is shown in the graph. In component A, there are no methanogenic bacteria; in component B, the concentration of methanogenic bacteria in the mixed bacterial agent is 1.55 × 10⁻⁶. 8 The concentration of methanogenic bacteria in the mixed bacterial agent added at C was 3.10 × 10⁻⁶ copies / mL. 8 The concentration of methanogenic bacteria in the mixed bacterial agent added to D was 4.65 × 10⁻⁶ / mL. 8 The concentration of methanogenic bacteria in the mixed bacterial agent added to E was 6.20 × 10⁻⁶ copies / mL. 8 The concentration of methanogenic bacteria in the mixed bacterial agent added at copy / mL and F was 9.30 × 10⁻⁶. 8 copy / mL; Figure 2 This is a graph showing the reduction and dehalogenation rate of the mixed microbial community simulating the actual remediation site in Example 4; In each diagram, TCE stands for trichloroethylene, DCE for cis-1,2-dichloroethylene, VC for vinyl chloride, ETH for ethylene, and CH4 for methane. Detailed Implementation
[0017] The present invention will be further described below with reference to specific embodiments, which will enable those skilled in the art to have a more comprehensive understanding of the invention, but will not limit the invention in any way. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.
[0018] Example 1: Preparation of liquid culture medium Composition of the liquid culture medium used to verify the reductive dehalogenation function: 1.0 g NaCl, 0.5 g MgCl2·6H2O, 0.2 g KH2PO4, 0.3 g NH4Cl, 0.3 g KCl, 0.015 g CaCl2·2H2O, 1 mL Se / W stock solution (6 mg / L Na2SeO3·5H2O, 8 mg / L Na2WO4·2H2O, 500 mg / L NaOH), 1 mL Trace element stock solution (1.5 g / L FeCl2·4H2O, 190 mg / L CoCl2·6H2O, 100 mg / L MnCl2·4H2O, 70 mg / L ZnCl2, 6 mg / L H3BO3, 36 mg / L Na2MoO4·2H2O, 24 mg / L NiCl2·6H2O, 2 mg / L CuCl2·2H2O), 2.292 g Tris ethanesulfonic acid was prepared by adding deionized water to a final volume of 1000 mL, boiling under nitrogen to remove oxygen, and adjusting the pH to 7-7.5. The culture medium was then evenly distributed into serum bottles, with 45 mL dispensed into each bottle. The bottles were sealed with rubber stoppers and sterilized at 121 °C for 20 min.
[0019] Example 2: Obtaining the anaerobic dehalogenation mixed microbial agent The mixed microbial agent used must contain at least dehalogenated Bacteroides ( Dehalococcoides mccartyi ), desulfuric vibrio ( Desulfovibrio desulfuricans Clostridium ( Clostridium sp. Petersonia mucosa ( Petrimonas mucosa Methanogens ( Methanobacterium formicicum ), methanogenic bacteria ( Methylocystis bryophila ).
[0020] The candidate strains are: Dehalogenated Cladosporium (ATCCBAA-2266), Desulfovibrio (KCTC5768), Clostridium (ATCC33052), Petersonia mucosa (CCAM323), Methanogens (ATCC33274), and Methanogens (NMDC20227199). Among them, Petersonia mucosa and Methanogens can be purchased from Ningbo Mingzhou Biotechnology Co., Ltd., while the others can be purchased from Gray Algae Biotechnology Co., Ltd.
[0021] The concentration range of each bacteria in the mixed bacterial agent is as follows: Dehalogenated Bacillus (9.68 × 10⁻⁶) 8 -2.82×10 9 copy / mL), desulfurized Vibrio (5.66×10 8 -6.21×10 8copy / mL), Clostridium (8.22×10) 7 -1.25×10 8 copy / mL), Petersonia mucosa (5.24×10 7 -3.43×10 8 copy / mL), Methanogens (7.25×10⁻⁶) 7 -1.38×10 8 (copy / mL) and methanogenic bacteria (2.24 × 10⁻⁶) 8 -4.29×10 8 (copy / mL), when using it, simply control the concentration of each bacterium in the mixed bacterial community to reach the above minimum limit, and ensure that the total bacterial community concentration in the pollutant is 1.97 × 10⁻⁶. 8 -4.48×10 8 Copy / mL is sufficient.
[0022] To facilitate subsequent experiments, this embodiment includes a gradient experiment with different concentrations of methanogenic bacteria. Specifically, the concentrations of all bacteria in the mixed bacterial community except for methanogenic bacteria are controlled to reach the aforementioned minimum limits, while methanogenic bacteria are added separately to prepare samples containing 1.55 × 10⁻⁶ methanogenic bacteria. 8 copy / mL (Group B), 3.10×10 8 copy / mL (Group C), 4.65×10 8 copy / mL (Group D), 6.20×10 8 copy / mL (Group E), 9.30×10 8 Mixed bacterial agent at a concentration of copy / mL (Group F).
[0023] Example 3 Functional Verification of Mixed Microbial Agents Add 5 mL of the mixed bacterial agent (Groups B-F) from Example 2 to each 45 mL bottle of liquid culture medium obtained in Example 1, so that the total bacterial concentration of the mixed bacterial agent in the culture medium is 3.07 × 10⁻⁶. 8 copy / mL, 3.22×10 8 copy / mL, 3.38×10 8 copy / mL, 3.53×10 8 copy / mL, 3.84×10 8Copy / mL. Then add TCE to achieve a concentration of 450 µmol / L (the added TCE corresponds to a chloride ion concentration of 1350 µmol / L, excluding the chloride ions in the culture medium itself); at the same time, add a carbon source and an electron donor substrate—lactic acid—to achieve a total lactic acid concentration of 10 mmol / L in the liquid culture medium, and incubate at a constant temperature of 30 °C in the dark.
[0024] Meanwhile, an anaerobic dehalogenated bacterial community without methanogenic bacteria was set up as a control group (Group A), so that the total bacterial community concentration in the culture medium was 2.91 × 10⁻⁶. 8 The procedure was the same as above. Changes in TCE and its products, as well as methane concentration, were periodically monitored in the six groups on days 0, 2, 5, 8, 10, 12, 15, and 17.
[0025] The results are as follows: (1) Mixed bacterial agent system without the addition of methanogenic bacteria Experimental Results: After 10 days of cultivation, trichloroethylene and intermediate products such as cis-1,2-dichloroethylene and vinyl chloride were all degraded into ethylene. The methane emission concentration was 388.88 µmol / L on day 8 and 695.31 µmol / L on day 10. Subsequently, as the system continued to develop, methane production continued to increase, reaching 1121.55 µmol / L on day 17. Figure 1 As shown in Figure A.
[0026] (2) Adding a mixed bacterial agent system containing different concentrations of methanogenic bacteria Experimental results: By adding the above mixed bacterial inoculum containing different concentrations of methanogenic bacteria, it was found that the total bacterial concentration in the system containing contaminants reached 3.22 × 10⁻⁶. 8 copy / mL (corresponding to a methanogenic bacteria concentration of 3.10 × 10⁻⁶ in the mixed bacterial culture) 8 At a concentration of (copy / mL), the dehalogenation rate was highest, achieving complete dehalogenation within 8 days. On day 8, the methane emission concentration was only 174.03 µmol / L, a 55.25% reduction compared to the 388.88 µmol / L in the group without methane-oxidizing bacteria on day 8. On day 10, the methane emission concentration was 361.33 µmol / L, a 48.03% reduction compared to the 695.31 µmol / L in the group without methane-oxidizing bacteria on day 10. On day 17, the methane emission concentration was 502.53 µmol / L, a 55.19% reduction compared to the 1121.55 µmol / L in the group without methane-oxidizing bacteria on day 17. Figure 1 As shown in C.
[0027] Other concentrations of mixed inoculum systems containing methanogenic bacteria can also reduce methane emissions, but all require 10 days to achieve complete dehalogenation. Therefore, group C, with a methanogenic bacteria concentration of 3.10 × 10⁻⁶, is preferred. 8 The optimal implementation scheme is a mixed bacterial agent with a concentration of [copy / mL], and the total bacterial concentration of the mixed bacterial agent in the system containing contaminants should be controlled to be at least 3.22 × 10⁻⁶. 8 copy / mL.
[0028] Example 4: Validation through simulation of actual repair site Using soil from the actual contaminated site as the substrate, tests confirmed that the soil contained some tetrachloroethylene and trichloroethylene. A simulation experiment was conducted with a water-to-soil mass ratio of 1:1. The molar ratio of tetrachloroethylene and trichloroethylene was artificially adjusted to 1:2, so that the total concentration of chloride ions in tetrachloroethylene and trichloroethylene was 1500 µmol / L. One group of mixed bacterial agents from Group C of Example 3 was added to the above mixed system, ensuring that the total bacterial concentration in the simulation experimental system reached at least 3.22 × 10⁻⁶. 8 copy / mL; simultaneously, a mixed bacterial agent of group A, which does not contain methanogenic bacteria, was added as a control group to make the total bacterial concentration in the simulated experimental system 2.91×10⁻⁶. 8 The concentration of the sample was 10 mmol / L to degrade tetrachloroethylene and trichloroethylene in the soil. A carbon source and an electron donor substrate—lactic acid—were added to achieve a total concentration of 10 mmol / L in the simulated experimental system for reductive dehalogenation experiments.
[0029] The results are as follows: Without the addition of methanogenic bacteria, the mixed bacterial agent in group A took 15 days to completely degrade tetrachloroethylene and trichloroethylene. However, with the addition of mixed bacterial agent in group C, tetrachloroethylene, trichloroethylene, and their intermediates were completely reduced and dehalogenated to ethylene within 12 days. Furthermore, the methane production decreased from 1272.21 µmol / L to 587.94 µmol / L after 12 days, and from 2126.51 µmol / L to 1075.63 µmol / L after 24 days. (Details are as follows...) Figure 2 As shown.
[0030] As can be seen from the above embodiments, the mixed bacterial agent with added specific concentrations of methanogenic bacteria provided in this application can more quickly and completely convert halogenated olefins, especially chlorinated olefins, into the non-toxic product ethylene, and can effectively reduce methane emissions, with methane emissions reduced by up to 55.25%.
[0031] The above embodiments are merely exemplary implementations used to illustrate the principles of the invention. However, the invention is not limited thereto. Those skilled in the art can make various improvements and modifications without departing from the essence of the invention, and these improvements and modifications also fall within the protection scope of the invention.
Claims
1. A mixed microbial agent for achieving efficient reductive dehalogenation and methane emission reduction, characterized in that: In an environment where water is used as a solvent, its effective active components include *Dehalogenated Coccidia*, *Desulfovibrio*, *Clostridium*, *Peter's Cells*, *Methanogens*, and 2.24 × 10⁻⁶. 8 -4.29×10 8 Methanogenic bacteria (copy / mL).
2. The mixed microbial agent for achieving efficient reduction dehalogenation and methane emission reduction according to claim 1, characterized in that: The concentration of dehalogenated *Streptococcus* strain in the mixed inoculum was 9.68 × 10⁻⁶. 8 -2.82×10 9 The concentration of *Vibrio desulfurans* was 5.66 × 10⁻⁶ copies / mL. 8 -6.21×10 8 The concentration of Clostridium difficile was 8.22 × 10⁻⁶ copies / mL. 7 -1.25×10 8 The concentration of Peterus mucosa was 5.24 × 10⁻⁶ copies / mL. 7 -3.43×10 8 The concentration of methanogenic bacteria was 7.25 × 10⁻⁶ copies / mL. 7 -1.38×10 8 copy / mL.
3. The mixed microbial agent for achieving efficient reductive dehalogenation and methane emission reduction according to claim 1 or 2, characterized in that: The mixed bacterial agent can be in liquid or solid form.