Microbial agent and method for efficiently degrading chlorinated organic matters and controlling methane emission

Abstract

The invention provides a microbial agent and method for cooperating with efficient degradation of chlorinated organics and controlling methane emission, and belongs to the technical field of chlorinated organics degradation, the method comprises the following steps: 1) mixing dechlorination flora with methanogens to obtain a microbial agent; (2) degrading chlorinated organic matters by using the microbial agent; the dechlorination flora contains bacillus subtilis of which the cell number accounts for more than 80%; the cell number ratio of the bacillus subtilis to the methanogens is (10-50): 1. By precisely regulating and controlling the ratio of the dechlorination bacteria to the methanogens, the degradation efficiency of COPs can be effectively improved, meanwhile, the generation amount of methane is reduced, and the synergistic interaction of pollution reduction and carbon reduction is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of chlorinated organic matter degradation, and in particular relates to a microbial agent and method for collaboratively degrading chlorinated organic matter with high efficiency and controlling methane emission. Background Art

[0002] Chlorinated organic compounds (COPs) are a class of persistent organic pollutants widely present in the environment. Due to their recalcitrance, bioaccumulation, and toxicity, they pose a serious threat to the ecological environment and human health and have become an environmental issue of global concern. Microbial-mediated reductive dechlorination is the main pathway for COPs degradation in anaerobic environments and is considered an economical, efficient, and environmentally friendly remediation technology. However, existing studies have shown that the reductive dechlorination process of COPs is often coupled with methanogenesis. COPs degradation is often accompanied by increased methane emissions, which not only reduces remediation efficiency but also creates a new environmental problem - greenhouse gas emissions, which is contrary to the current global strategic goal of "reducing pollution and carbon emissions."

[0003] In the coupled dechlorination and methanogenesis system, dechlorinating bacteria and methanogens are key functional groups driving pollutant degradation and energy recycling. A complex interaction exists between the two, characterized by both mutual benefits and competitive dynamics. On the one hand, dechlorinating bacteria, through reductive dehalogenation, convert highly chlorinated organic matter into low-chlorinated or even unchlorinated products, providing methanogens with usable substrates (such as acetic acid and H₂). The H₂ and cofactors produced by methanogens can also feed back to the dechlorinating bacteria, enhancing dechlorination activity and forming a potential metabolic symbiosis. On the other hand, dechlorinating bacteria and methanogens may compete for limited electron donors and carbon sources, leading to niche competition. This complex relationship determines the efficiency and stability of the coupled dechlorination and methanogenesis process and shapes the functional landscape of anaerobic ecosystems. However, existing technologies lack a deep understanding of the interaction mechanisms between dechlorinating bacteria and methanogens, and lack effective methods to regulate this coupling relationship. This makes it difficult for bioremediation technologies to achieve both efficient degradation of COPs and control of methane emissions.

[0004] Ecological theory indicates that population ratio is an important factor affecting the structure and function of microbial communities. By regulating the initial inoculation ratio of key functional groups, the interaction pattern and functional output of the community can be significantly changed. Especially in anaerobic environments with limited resources, the initial inoculation ratio not only affects the formation path of the community structure, but also determines the construction of metabolic networks and the process of niche differentiation among functional bacterial groups, thereby affecting the functional expression of the entire system. However, existing bioremediation technologies rarely optimize the dechlorination and methanogenesis coupling process from the perspective of regulating bacterial population ratios to achieve a strategy for the coordinated control of efficient degradation and low methane emissions.

[0005] Existing bioremediation technologies, such as enhanced anaerobic bioremediation technology, mainly stimulate local microbial activity and promote COPs degradation by adding electron donors, nutrients and other means. For example, Chinese patent CN117358748B states that "under an anaerobic environment, by adding at least one of electron donors, electron shuttles and electron flow disruptors to the soil, the entire electron transfer process is precisely regulated, so that the reduction and dechlorination of indigenous microorganisms and the weakening of methanogen metabolism are achieved, thereby achieving the remediation of organochlorine-contaminated soil and methane emission reduction." In addition, some studies have attempted to reduce methane emissions by adding methane inhibitors, but this may also inhibit the activity of dechlorinating bacteria and reduce the efficiency of COPs degradation. The use of chemical inhibitors may also bring the risk of secondary pollution. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a microbial agent and method for synergistically degrading chlorinated organic matter and controlling methane emissions. By precisely controlling the ratio of dechlorinating bacteria to methanogens, the degradation efficiency of COPs can be effectively improved while reducing the amount of methane generated, thereby achieving synergistic efficiency in "pollution reduction and carbon reduction."

[0007] The present invention provides a method for synergistically degrading chlorinated organic compounds and controlling methane emissions, comprising the following steps:

[0008] 1) Mixing dechlorinating bacteria with methanogens to obtain a microbial agent;

[0009] 2) using the microbial agent to degrade chlorinated organic matter;

[0010] The dechlorinating bacteria group contains more than 80% of Bacillus subtilis cells that have the function of degrading chlorinated organic matter;

[0011] The cell number ratio of the Bacillus subtilis to the methanogen is (10-50):1.

[0012] Preferably, the cell number ratio of the Bacillus subtilis to the methanogen is (18-25):1.

[0013] Preferably, the cell number ratio of Bacillus subtilis to methanogens is 21:1.

[0014] Preferably, the dechlorinating bacteria are obtained by anaerobic enrichment and domestication of samples contaminated by chlorinated organic matter.

[0015] Preferably, the chlorinated organic matter is selected from one or more of hexachlorocyclohexane, polychlorinated biphenyls, polychlorinated vinyls, polychlorinated ethanes and chlorinated benzene compounds.

[0016] Preferably, the degradation of the chlorinated organic matter in step 2) is carried out under anaerobic conditions, the degradation temperature is 25-35° C., the pH value of the degradation system is 6.5-7.5, and the degradation time is 10-30 days.

[0017] The present invention provides a microbial agent for synergistically degrading chlorinated organic matter and controlling methane emissions, comprising a dechlorinating bacterial group and a methanogen; the dechlorinating bacterial group contains more than 80% of Bacillus subtilis with the function of degrading chlorinated organic matter, and the cell number ratio of the Bacillus subtilis to the methanogen is (10-50):1.

[0018] Preferably, the microbial agent further comprises nutrients and protective agents.

[0019] The present invention provides a method for repairing chlorinated organic pollution, wherein the microbial agent is applied to the object to be repaired, and an anaerobic environment is maintained to carry out the repair.

[0020] Preferably, the object to be repaired is soil contaminated by chlorinated organic compounds, sediment contaminated by chlorinated organic compounds, or water contaminated by chlorinated organic compounds; and the method of maintaining an anaerobic environment includes flooding or film covering.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The method for synergistically degrading chlorinated organic matter and controlling methane emissions provided by the present invention achieves the optimal effect of synergistically degrading chlorinated organic matter and controlling methane emissions by precisely regulating the ratio of Bacillus subtilis to methanogens.

[0023] In the present invention, the dechlorinating bacteria group enriched anaerobically is primarily Bacillus subtilis, which plays a key role in the anaerobic dechlorination process, efficiently reducing and dechlorinating various chlorinated organic compounds (such as lindane to chlorobenzene), and further metabolizing them to produce small-molecule organic acids such as acetic acid and butyric acid. Methanosarcina methanogens can utilize the intermediates produced during the dechlorination process (such as chlorobenzene, acetic acid, and H2) to produce methanogens, achieving carbon recycling. When the two coexist in a specific ratio, they can form complementary advantages, improving carbon source utilization efficiency and the thoroughness of pollutant degradation.

[0024] The present invention optimizes the distribution of carbon metabolic flows by controlling the ratio of Bacillus subtilis to methanogens in the dechlorinating bacterial community, so that more carbon sources are used for the dechlorination process rather than methane production. Bacillus subtilis is dominant in number and occupies a dominant position in the competition for carbon sources, which limits the carbon source utilization of methanogens, thereby reducing methane emissions. When working together, methanogens can metabolize certain inhibitory intermediates produced in the dechlorination process, or provide certain growth factors, thereby enhancing the dechlorination efficiency of Bacillus subtilis. The embodiments of the present invention record that, compared with a single bacterial community or a mixed bacterial system with unoptimized ratios, the 30-day lindane degradation rate of the method of the present invention is increased to 84%, and there is no risk of increased methane emissions.

[0025] Based on a thorough analysis of the interaction mechanisms, this paper defines the optimal bacterial population ratios and culture conditions, providing clear parameter guidance for the optimization and application of bioremediation technology, with good operability and controllability. The technical solutions provided by this invention enable the rapid construction of efficient and low-carbon bioremediation systems.

[0026] The present invention utilizes the natural degradation ability of microorganisms, does not need to add additional chemicals, reduces the risk of secondary pollution, and has good environmental friendliness. The method described in the present invention can be applied to the bioremediation of anaerobic environments (such as soil, sediment, groundwater, etc.) polluted by multiple chlorinated organic compounds, is particularly suitable for typical pollution scenes such as farmland soil and wetland sediments, and has a wide range of applications and environmental benefits. The application and promotion of the present invention will contribute to solving the chlorinated organic compound pollution problem worldwide, while responding to the strategic goal of global "pollution reduction and carbon reduction", with important environmental and social significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a process flow chart for the coordinated methane emission reduction process for soil remediation contaminated by chlorinated organic compounds according to the present invention;

[0028] Figure 2 The microbial community structure of the paddy soil sample before and after anaerobic enrichment and acclimation in Example 1; wherein a is the community structure of anaerobic enrichment and acclimation cultures of different generations, and b is the community structure of the enrichment culture after stabilization. DETAILED DESCRIPTION

[0029] The present invention provides a method for synergistically degrading chlorinated organic matter and controlling methane emissions, comprising the following steps: 1) mixing a dechlorinating bacterial group with a methanogen to obtain a microbial agent; 2) using the microbial agent to degrade the chlorinated organic matter; more than 80% of the cells of the dechlorinating bacterial group are Bacillus subtilis having the function of degrading chlorinated organic matter; and the cell number ratio of the Bacillus subtilis to the methanogen is (10-50):1.

[0030] In the present invention, a microbial inoculum is obtained by mixing dechlorinating bacteria with methanogens. The dechlorinating bacteria are obtained by anaerobic enrichment and acclimation of a sample contaminated with chlorinated organic compounds. The chlorinated organic compounds are preferably selected from one or more of hexachlorocyclohexane, polychlorinated biphenyls (PCBs), polychlorinated vinyls (PCEs), polychlorinated ethanes (PCEs), and chlorinated benzene compounds. In a specific embodiment of the present invention, the chlorinated organic compound is γ-hexachlorocyclohexane, commonly known as lindane. First, a soil sample is collected. The soil sample is preferably a sample of soil long-term contaminated with chlorinated organic compounds, more preferably paddy soil contaminated with chlorinated organic compounds. After obtaining the soil sample, the present invention performs anaerobic enrichment and acclimation; the anaerobic enrichment and acclimation is preferably performed by mixing the soil sample with the culture medium and then culturing in the dark under anaerobism; the anaerobic culture is preferably performed in an anaerobic incubator with shaking, and the temperature of the anaerobic culture is preferably 29-31°C, more preferably 30°C; the mass volume ratio of the soil sample to the culture medium is 1g: (5-15) ml, more preferably 1g: (8-12) ml, and more preferably 1g: 10 ml. In the present invention, the culture medium contains chlorinated organic matter, and the chlorinated organic matter is preferably lindane. The concentration of lindane in the culture medium is preferably 40-60 mg / L, more preferably 45-55 mg / L, and more preferably 50 mg / L. In the present invention, the culture medium uses water as a solvent and also includes a basic salt solution, a trace element solution, a vitamin solution, a reducing agent, a sodium bicarbonate buffer and an electron donor; the electron donor is preferably sodium acetate. In the process of the anaerobic enrichment and acclimation of the present invention, sampling is performed at fixed intervals, preferably every 8 to 12 days, more preferably every 10 days; after sampling, the lindane concentration and the microbial community structure are measured. In the present invention, when the lindane degradation rate is stable and the microbial community structure no longer changes significantly, the anaerobic enrichment and acclimation is terminated; the total time of the anaerobic enrichment and acclimation is usually 20 to 60 days. In the present invention, the lindane degradation rate is stable, preferably the degradation rate is stable at more than 50%, and the degradation rate changes by <5% after passage; the microbial community is preferably dominated by Bacillus subtilus in the genus Bacillus, and the cell number accounts for more than 80%, preferably 85% to 86%.

[0031] After obtaining a dechlorinating bacterial population, the present invention mixes the dechlorinating bacterial population with methanogens to produce a microbial agent; the methanogens are preferably the model methanogen strain Methanosarcina barkeri (DSM 800). The methanogens are preferably cultured according to the DSMZ culture medium manual. The culture medium for culturing the methanogens has the same composition as the dechlorinating bacterial population, differing only in that it does not contain lindane. The cell number ratio of Bacillus subtilis to methanogens is (10-50):1, preferably (18-25):1, and more preferably 21:1. The cell number ratio of Bacillus subtilis to methanogens specified in the present invention is optimized based on extensive experiments and can achieve optimal dechlorination efficiency and methane control effects. Excessively high or low ratios can result in decreased dechlorination efficiency or increased methane emissions. Precise control of the bacterial population ratio is one of the core innovations of the present invention. By regulating the bacterial population ratio, the interaction between the dechlorinating bacteria and methanogens can be effectively balanced, fully maximizing their synergistic effects while suppressing excessive methane production.

[0032] In the present invention, the dechlorinating bacteria and methanogens obtained through anaerobic enrichment and acclimation are preferably centrifuged and washed to remove residual culture medium, and the cells are collected. The cell concentrations of the two bacterial communities are then measured, and the cells are mixed according to the cell number ratio defined above to obtain a microbial inoculum. In the present invention, the initial inoculum ratio of Bacillus subtilis to methanogens is determined and controlled by methods such as cell counting, fluorescent quantitative PCR, or high-throughput sequencing. Cell counting is a direct and simple method, while fluorescent quantitative PCR and high-throughput sequencing methods can more accurately quantify bacterial abundance and monitor changes in community structure.

[0033] After obtaining the composite bacterial consortium, the present invention uses the microbial agent to degrade chlorinated organic matter. In the present invention, the degradation of chlorinated organic matter is carried out under anaerobic conditions. The degradation temperature is preferably 25-35°C, the pH of the degradation system is preferably 6.5-7.5, and the degradation time is preferably 10-30 days. In the present invention, nutrients are preferably added during the degradation process. Anaerobic conditions are key to ensuring the smooth progress of the reductive dechlorination process. Appropriate nutrients can provide the microorganisms with the energy and substances required for growth and metabolism, and optimized temperature and pH can maintain microbial enzyme activity, thereby ensuring efficient pollutant degradation. During the degradation process, the present invention preferably monitors and controls the degradation of chlorinated organic matter in the culture system (including the concentration of intermediates and final products), methane production, microbial community structure (abundance of Bacillus and Methanosarcina), and changes in metabolites. Monitoring indicators in the present invention include, but are not limited to, chlorinated organic matter concentration, methane concentration, microbial cell density, abundance of key functional genes (such as rdhA and mcrA), and the types and contents of metabolic intermediates. The present invention preferably further optimizes the culture conditions (such as carbon source type, pH, temperature, electron donor / acceptor, etc.) and the proportion of the bacterial community based on the monitoring results, and maintains the cell number ratio of Bacillus subtilis and methanogens within the ratio range defined by the present invention to achieve the best degradation effect and methane control effect. For example, when it is monitored that the methane production has an increasing trend, the proportion of methanogens can be appropriately adjusted, or the carbon source type can be optimized to inhibit methane production. In the present invention, molecular biology techniques such as metagenomics, macrotranscriptomics or targeted gene amplification sequencing are used to monitor the structure of the microbial community. Metagenomics and macrotranscriptomics can comprehensively analyze the composition, functional potential and gene expression of the microbial community, while targeted gene amplification sequencing can quickly quantify the abundance changes of specific functional bacterial communities.

[0034] The present invention also provides a microbial agent for synergistically degrading chlorinated organic matter and controlling methane emissions, including a dechlorinating bacterial group and a methanogen; the dechlorinating bacterial group contains Bacillus subtilis with the function of degrading chlorinated organic matter, accounting for more than 80% of the cell number, and the cell number ratio of the Bacillus subtilis to the methanogen is (10-50):1, preferably (18-25):1, and more preferably 21:1. The present invention does not specifically limit the specific community structure of the dechlorinating bacterial group, and provides a Bacillus subtilis strain that can efficiently degrade chlorinated organic matter, or an efficient dechlorinating bacterial group containing Bacillus subtilis as the main functional strain. In the present invention, the dechlorinating bacterial group is preferably obtained by anaerobic enrichment and domestication, or obtained by artificially compounding Bacillus subtilis with a clear dechlorination function with other bacterial species. The method for obtaining the dechlorinating bacterial group by anaerobic enrichment and domestication is described above and will not be repeated here. In the present invention, the microbial inoculant further comprises nutrients and protective agents. In the present invention, the nutrients include a carbon source, a nitrogen source, vitamins, and trace elements. The carbon source is preferably acetic acid, sodium acetate, lactic acid, or a combination thereof, more preferably sodium acetate. The nitrogen source is preferably yeast extract. In the present invention, the protective agent is preferably glycerol, trehalose, and / or skim milk powder. The addition of nutrients in the present invention can provide the microorganisms in the microbial inoculant with energy and substances to initiate metabolism, thereby improving the repair effect of the microbial inoculant. The addition of protective agents can improve the stability of the microbial inoculant during storage and transportation, thereby extending the shelf life of the inoculant. In the present invention, the microbial inoculant is preferably a liquid inoculant or a solid inoculant, and the solid inoculant preferably includes a freeze-dried inoculant. In the present invention, the liquid inoculant can be used directly and is easy to operate. The solid inoculant is convenient to store and transport and needs to be revived before use.

[0035] The present invention provides a method for remediating chlorinated organic contamination, comprising applying the microbial agent to the object to be remediated, maintaining an anaerobic environment, and performing remediation. In the present invention, the object to be remediated is preferably chlorinated organic contaminated soil, chlorinated organic contaminated sediment, or chlorinated organic contaminated water. The method for maintaining the anaerobic environment preferably includes flooding or film covering; the film covering is preferably an airtight film. The present invention preferably also includes a step of pretreating the object to be remediated prior to remediation. The pretreatment includes adjusting the soil moisture content to saturation, adjusting the pH to 6.5-7.5, or adding nutrients to promote the growth and metabolic activity of the microbial agent. In the present invention, the pretreatment can improve the physical and chemical properties of the soil or sediment and enhance the remediation effect of the microbial agent. In the present invention, the microbial agent can be applied in a variety of ways, including but not limited to direct mixing, perfusion, or ex situ remediation using a bioreactor. Direct mixing and perfusion are suitable for in-situ remediation, while bioreactors are suitable for ex situ remediation. The present invention preferably selects the appropriate remediation method based on the specific conditions of the contaminated site.

[0036] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] (1) Source and culture of bacteria:

[0039] Dechlorinating bacteria: Paddy soil at a depth of 15-30 cm in Baifeng'ao, Taizhou City, Zhejiang Province, was used as the inoculum source. This soil had long been contaminated with chlorinated organic pesticides. Anaerobic enrichment and acclimation cultivation was performed using 50 mg / L lindane (γ-HCH) as the sole electron acceptor, with shaking in a medium containing the following ingredients.

[0040] Culture medium composition:

[0041] Basic salt solution (g / L): NH4Cl 1.0, KH2PO4 0.2, MgCl2·6H2O 0.1, CaCl2·2H2O 0.01.

[0042] Trace element solution (mL / L): 10 (containing, g / L: EDTA 50, FeSO4·7H2O 2, ZnSO4·7H2O 1, MnCl2·4H2O 0.5, H3BO3 0.05, CoCl2·6H2O 0.1, CuCl2·2H2O 0.03, NiCl2·6H2O 0.02, Na2MoO4·2H2O 0.03).

[0043] Vitamin solution (mL / L): 10 (contains, mg / L: biotin 2, folic acid 2, pyridoxine hydrochloride 10, thiamine hydrochloride 5, riboflavin 5, niacin 5, calcium pantothenate 5, vitamin B12 0.1, para-aminobenzoic acid 5, lipoic acid 5).

[0044] Reducing agent: 0.5 mM sodium sulfide (Na2S·9H2O).

[0045] Sodium bicarbonate buffer (84 g / L NaHCO3): Adjust the pH to 7.0-7.2.

[0046] Electron donor: 5 mM sodium acetate.

[0047] Add deionized water to 1 L.

[0048] Soil samples were mixed with culture medium at a ratio of 5g:50ml and incubated in an anaerobic incubator at 30°C in the dark for 30 days. Samples were collected every 10 days to measure lindane concentration and microbial community structure. Enrichment culture was terminated when the lindane degradation rate stabilized and the microbial community structure no longer changed significantly, resulting in a highly efficient dechlorinating bacterial community dominated by Bacillus.

[0049] Original soil sample community structure:

[0050] The bacterial abundance was 69.55%, dominated by acidogenic fermentative bacteria; the archaeal abundance was 30.45%, dominated by methanogens. The most abundant genera included: (1) dechlorinating bacteria: Dehalococcoides; (2) hydrogen-producing and acetogenic bacteria: Candidatus Cloacamonas, Aminivibrio, Sphaerochaeta, Desulfovibrio; and (3) corrinoid-producing bacteria: Methanobacterium, Methanothrix, Methanosarcina.

[0051] After 10 days of acclimation, the enriched culture showed sustainable γ-HCH degradation ability independent of oxygen, with a γ-HCH degradation rate of approximately 59.7% in 10 days, compared with sterile culture (32.6% γ-HCH degradation) and pure culture (30.9% γ-HCH degradation).

[0052] After 30 days of acclimation, a stable dechlorinating bacterial community was obtained. High-throughput sequencing analysis (Illumina MiSeq, 16S rRNA gene V4 region) revealed the following composition of the acclimated dechlorinating bacterial community: Bacillus subtilis: 85.57% ± 2.3% (relative abundance), Unassigned: 6.21% ± 1.1%, Lysinibacillus spp.: 3.85% ± 0.9%, and other: <5% (total). This composition was verified by five independent acclimation experiments, with a coefficient of variation of <15% (based on Bray-Curtis distance).

[0053] During the entire anaerobic enrichment acclimation process, the dominant Bacillus could grow stably under lindane stress, and its cell copy number increased by 36.68 times from the initial 4.08e 6 copies ml -1 Grows to a final value of 1.49e 8 copies ml -1 .

[0054] Methanogenic bacteria: The model methanogenic strain Methanosarcina barkeri (DSM 800) was selected. Culture was performed according to the DSMZ culture medium manual. The medium composition was the same as that for the dechlorinating bacteria described above, but without lindane.

[0055] (2) Construction of mixed bacteria system:

[0056] The enriched dechlorinating bacterial colony dominated by Bacillus species is centrifuged and washed to remove residual culture medium. A pure culture of Methanosarcina barkeri (e.g., DSM 800) is also cultured and similarly centrifuged and washed. The cell concentrations of the two bacterial colonies are determined using quantitative PCR.

[0057] Experimental grouping (proportional gradient setting): According to the cell number ratio, the dechlorinating bacteria group and the Methanosarcina barkeri strain were mixed in the following proportions to prepare mixed bacterial suspensions of different proportions:

[0058] a) Single bacteria group (Bac): only dechlorinating bacteria were inoculated.

[0059] b) Mix-Low group: Bacillus:Methanosarcina in the dechlorinating bacteria group = 3:1.

[0060] c) Mix-Opt: Bacillus:Methanosarcina in the dechlorinating bacteria group = 21:1.

[0061] d) Mix-High group: Bacillus:Methanosarcina in the dechlorinating bacteria group = 126:1.

[0062] e) Single-strain group (Met): only the Methanosarcina strain was inoculated.

[0063] f) Blank control group (CK): no bacterial inoculation.

[0064] Precise control of the ratio is the key, and the concentration of the bacterial solution can be adjusted multiple times to ensure the final mixing ratio is accurate.

[0065] (3) Anaerobic culture and remediation application:

[0066] Microcosm construction: 5 g of lindane-contaminated soil (initial concentration 50 mg / kg dry soil) was sieved (2 mm pore size) and placed in a 30 mL serum bottle. 50 mL of sterile water was added and the bottle was sealed with a butyl rubber stopper and an aluminum cap. The bottle was vacuumed and filled with nitrogen three times to simulate flooded anaerobic conditions.

[0067] Inoculation and culture: The serum bottles of each treatment group were inoculated with the above bacterial suspensions in different proportions (the inoculation volume ensured that the initial Bacillus cell count was consistent, approximately 1×10 7 cells / g dry soil). An equal amount of sterile water was added to the CK group. Lindane was added to all serum bottles to a final concentration of 10 mg / kg dry soil, and 5 mM sodium acetate was added as a carbon source. Cultures were placed in an anaerobic incubator at 30°C in the dark for 30 days.

[0068] (4) Monitoring: Samples were collected on days 0, 3, 5, 10, 20, and 30 of culture to measure the following indicators:

[0069] i.γ-HCH concentration: measured by gas chromatography-mass spectrometry (GC-MS).

[0070] ii. Methane production: measured using a gas chromatograph (FID detector).

[0071] iii. Bacillus and Methanosarcina cell copy number: Quantitative PCR was performed using specific primers. For Bacillus, the primers were Bacillus-Forward (5′-AAGGGCTTCACCATCTTGTAT-3′) and Bacillus-Reverse (5′-TTACATGGTGTTGGGGCATCG-3′); for Methanosarcina, the primers were mcrA-mod-F (GGYGGTGTMGGDTTCACMCARTA) and mcrA-R (CGTTCATBGCGTAGTTVGGRTAGT).

[0072] iv. Metabolites: Non-targeted metabolomics analysis was performed using liquid chromatography-mass spectrometry (LC-MS) to identify intermediate metabolites. The repair effects of different groups are shown in Table 1.

[0073] Table 1 Lindane degradation rate and methane accumulation in different treatment groups

[0074] Group Bacillus:Methanosarcina Lindane degradation rate (%) Methane accumulation (uM) CK / 9.0±0.5 1.9±0.5 Bac 1:0 50.4±4.2 1.8±0.7 Mix-Low 3:1 54.6±3.4 3.6±0.3 Mix-Opt 21:1 84.1±1.1 2.1±0.4 Mix-High 126:1 48.7±2.0 10.4±0.9 Met 0:1 33.8±5.3 9.3±0.6

[0075] The experimental results clearly demonstrated that a mixed bacterial system with a Bacillus to Methanosarcina cell ratio of approximately 21:1 (Mix-Opt group) exhibited the best synergistic effect in the remediation of lindane-contaminated soil, achieving the highest lindane degradation efficiency (84.1%), significantly higher than the single bacterial groups (Bac group 50.4%, Met group 33.8%) and mixed bacterial groups with other ratios (Mix-Low: 54.6%, Mix-High: 48.7%). Furthermore, the methane accumulation in the Mix-Opt group (2.1 μM) was comparable to that in the Bac group (1.8 μM) and the blank control group (1.9 μM), indicating that at this optimal ratio, methane emissions were effectively controlled and there was no risk of increased methane emissions. In contrast, methane emissions were significantly increased in the Met group (9.3 μM) and the Mix-High group (10.4 μM).

[0076] The experimental data of this example strongly verified the effectiveness and superiority of the technical solution of the present invention, and for the first time clarified that by precisely controlling the ratio of dechlorinating bacteria to methanogens, the degradation efficiency of COPs can be effectively improved while reducing the amount of methane generated, thereby achieving synergistic efficiency in "pollution reduction and carbon reduction."

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for efficiently degrading chlorinated organic compounds and controlling methane emissions, characterized in that: The following steps are involved: 1) Mixing dechlorinating bacteria with methanogens to obtain a microbial agent; 2) using the microbial agent to degrade chlorinated organic matter; The dechlorinating bacteria group contains more than 80% of Bacillus subtilis cells that have the function of degrading chlorinated organic matter; The cell number ratio of the Bacillus subtilis to the methanogen is (10-50):

1.

2. The method according to claim 1, characterized in that The cell number ratio of the Bacillus subtilis to the methanogen is (18-25):

1.

3. The method according to claim 2, characterized in that The cell number ratio of the Bacillus subtilis to the methanogens is 21:

1.

4. The method according to claim 1, wherein The dechlorinating bacteria group is obtained by anaerobic enrichment and domestication of a sample contaminated by chlorinated organic matter.

5. The method according to claim 1 or 4, characterized in that The chlorinated organic matter is selected from one or more of hexachlorocyclohexane, polychlorinated biphenyls, polychlorinated vinyls, polychlorinated ethanes and chlorinated benzene compounds.

6. The method according to claim 1, wherein Step 2) The degradation of the chlorinated organic matter is carried out under anaerobic conditions, the degradation temperature is 25-35° C., the pH value of the degradation system is 6.5-7.5, and the degradation time is 10-30 days.

7. A microbial agent for synergistically degrading chlorinated organic compounds and controlling methane emissions, characterized in that: The invention comprises a dechlorinating bacteria group and a methanogen; the dechlorinating bacteria group contains more than 80% of Bacillus subtilis having the function of degrading chlorinated organic matter, and the cell number ratio of the Bacillus subtilis to the methanogen is (10-50):

1.

8. The microbial agent according to claim 7, characterized in that The microbial agent also contains nutrients and protective agents.

9. A method for repairing chlorinated organic pollution, characterized in that: The microbial agent according to claim 7 or 8 is applied to the object to be repaired, and an anaerobic environment is maintained to carry out the repair.

10. The method according to claim 9, characterized in that The object to be repaired is soil contaminated by chlorinated organic compounds, sediment contaminated by chlorinated organic compounds, or water body contaminated by chlorinated organic compounds; the method of maintaining the anaerobic environment includes flooding or covering with a film.

Images