Method for culturing aerobic methanogenic microorganisms in mangrove sediment

Abstract

This invention provides a method for culturing aerobic methanogenic microorganisms in mangrove sediments, belonging to the field of microbial technology. The method comprises five steps: culture medium preparation, sample pretreatment, mud system preparation, aerobic environment construction, and isothermal incubation. The culture medium contains basic salts, macroelements, microelements, and vitamins adapted to the brackish water environment of mangroves. By constructing a stable aerobic culture system, the efficient enrichment of aerobic methanogenic microorganisms in mangrove sediments is achieved. This invention is specifically adapted to the environmental characteristics of mangrove sediments, filling the gap in aerobic methanogenic microorganism enrichment and cultivation technology in mangrove ecosystems. It can stably achieve the cultivation and enrichment of aerobic methanogenic microorganisms, providing reliable technical support for elucidating the aerobic methanogenesis process in coastal mangrove wetlands, isolating and purifying aerobic methanogenic functional microorganisms, and expanding the methanogenic microbial resources of mangrove ecosystems.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a method for culturing aerobic methanogenic microorganisms from mangrove sediments. Background Technology

[0002] Methane is a significant greenhouse gas, with a greenhouse effect 28 times greater than that of carbon dioxide, making it a key factor influencing global climate change. In recent years, atmospheric methane levels have been rising steadily. Controlling and reducing methane emissions is crucial to preventing global warming and related climate disasters.

[0003] Atmospheric methane is broadly classified into anthropogenic and natural sources, with wetlands being the primary natural source. Methane emitted by wetland ecosystems is mainly produced by microbial metabolism. The classic methanogenesis process involves methanogenic archaea synthesizing methane using small organic or inorganic molecules under strictly anaerobic conditions. With further research, researchers have discovered aerobic methanogenesis processes in oceans and lakes. In these processes, microorganisms utilize methyl compounds such as methyl sulfides, methylamine, and methyl phosphates to generate methane in an aerobic environment. Currently, significant methane production has been detected in oxygen-rich waters across various ocean regions globally, primarily originating from aerobic methanogenesis using methyl phosphates and methyl sulfides.

[0004] Mangrove wetlands are vast "blue carbon" reservoirs in the land-sea interface region and hotspots for greenhouse gas emissions such as methane. The material cycling and microbial community structure of mangrove sediments exhibit significant uniqueness. Current research on microbial-driven methanogenesis primarily focuses on anaerobic methanogenesis, while the processes and mechanisms of aerobic methanogenesis in this ecosystem remain unclear. The main bottleneck lies in the lack of targeted enrichment and cultivation techniques for aerobic methanogenic microorganisms. Existing enrichment and cultivation techniques for aerobic methanogenic microorganisms mainly target marine and lake environmental samples, using methyl phosphate as a single substrate. No reports have been published on enrichment and cultivation techniques for aerobic methanogenic microorganisms specific to mangrove sediment samples, adapted to the brackish water environment of mangroves, and utilizing multiple methyl-based substrates. This severely restricts research on aerobic methanogenesis processes in mangrove wetlands and the discovery of functional microorganisms.

[0005] Therefore, a method for culturing aerobic methanogenic microorganisms in mangrove sediments is proposed. Summary of the Invention

[0006] The purpose of this invention is to address the gaps and deficiencies in existing technologies by providing a method for cultivating aerobic methanogenic microorganisms in mangrove sediments. This method is adapted to the characteristics of the brackish water environment of mangroves and can utilize various methyl compounds as substrates to stably and efficiently enrich aerobic methanogenic microorganisms in mangrove sediments, providing reliable technical support for elucidating the aerobic methanogenesis process in mangrove wetlands.

[0007] The specific technical solution is as follows: A method for culturing aerobic methanogenic microorganisms in mangrove sediments includes the following steps: (1) Culture medium preparation: Prepare a culture medium suitable for the brackish water environment of mangroves. The culture medium includes basic salt components, macro element components, trace element components and vitamin components. Adjust the pH of the culture medium to neutral. (2) Sample pretreatment: Take surface mangrove sediment samples and remove large solid impurities from the samples; (3) Preparation of mud system: Mix the pretreated sediment sample with the culture medium obtained in step (1) according to the preset solid-liquid ratio and stir until a uniform mud system is formed; (4) Construction of aerobic culture system: The mud system is transferred to a sterilized culture container and sealed. The headspace of the culture container is replaced with gas to construct and maintain an aerobic culture environment. (5) Enrichment culture: The sealed culture container is placed in a constant temperature and light-proof environment for culture to complete the enrichment of aerobic methanogenic microorganisms in mangrove sediments.

[0008] This scheme establishes a complete culture process adapted to the target habitat, fully simulating the basic characteristics of the original environment of the sample. It can build a stable and suitable growth system for the target group, realize the stable proliferation and enrichment of the target group, fill the gap in the culture technology of the target group in the corresponding habitat, and provide a reliable technical foundation for subsequent related research and applications.

[0009] The above-mentioned method for culturing aerobic methanogenic microorganisms in mangrove sediments includes: In step (1), the basic salt component is sterilized by high temperature and high pressure. The macro-element component, trace element component, and vitamin component are respectively prepared into sterile mother liquor. After the basic salt solution is cooled to room temperature, an appropriate amount of macro-element component, trace element component, and vitamin component are added, and the pH is adjusted to neutral. In step (2), the large solid impurities are gravel and fragments of branches and leaves; In step (3), the sediment sample is measured by wet weight; In step (4), the culture container is a sterile anaerobic bottle, sealed with a sterile rubber stopper and an aluminum cap; the gas replacement operation is as follows: after evacuating the headspace of the anaerobic bottle, a mixture of oxygen and monofluoromethane is injected, and the evacuation and injection operations are repeated to fill the headspace of the anaerobic bottle with a mixture of oxygen and monofluoromethane and restore the pressure inside the bottle to normal pressure.

[0010] This scheme clarifies the key operational details of each step in the culture process, which can fully guarantee the stability of the sterile environment and growth conditions of the culture system, avoid contamination by other microorganisms and environmental fluctuations during operation, further improve the stability and reproducibility of the culture system, and ensure that the target population can grow normally under stable and controllable conditions.

[0011] The above-mentioned method for cultivating aerobic methanogenic microorganisms in mangrove sediments, wherein in step (1), the final concentration composition of the basic salt components is as follows: NaCl 400mM~550mM, MgCl2·6H2O 20mM~35mM, CaCl2·2H2O 5mM~15mM, KCl 5mM~15mM, and MgSO4·7H2O 2mM~4mM.

[0012] The proposed scheme provides a wide range of basic salt components that fully simulate the ionic composition and osmotic pressure characteristics of the native environments of samples from different regions. This avoids growth inhibition of target groups due to sudden environmental changes or unsuitable ionic conditions, ensuring normal intracellular physiological and biochemical activities of the target groups and significantly improving the adaptability of the culture system to samples from different sources. The above-mentioned method for cultivating aerobic methanogenic microorganisms in mangrove sediments, wherein in step (1), the final concentration of the macro-element components is: (NH4)2SO4 is 300μM~500μM, and NaH2PO4 with pH 7.5 is 30μM~80μM.

[0013] This scheme clarifies the compatibility range of macronutrient components, which can provide sufficient basic nutrient supply for the growth and reproduction of the target group, meet the basic material requirements for cell synthesis and proliferation of the target group, and provide the materials and energy required for the normal growth of the target group.

[0014] The above-mentioned method for cultivating aerobic methanogenic microorganisms in mangrove sediments, wherein in step (1), the final concentration composition of the trace element components is as follows: FeCl3·6H2O is 100nM~150nM, MnCl2·4H2O is 5nM~15nM, ZnSO4·7H2O is 500pM~1000pM, CoCl2·6H2O is 300pM~800pM, Na2MoO4·2H2O is 200pM~500pM, Na2SeO3 is 0.5nM~2nM, and NiCl2·6H2O is 0.5nM~2nM.

[0015] This approach, by limiting the range of micronutrient compatibility, can provide essential cofactors for the synthesis of key enzymes in the metabolic process of the target group, ensuring the normal operation of the core metabolic pathways of the target group, while maintaining normal intracellular physiological responses, and effectively enhancing the metabolic activity and growth capacity of the target group.

[0016] The above-mentioned method for culturing aerobic methanogenic microorganisms in mangrove sediments, wherein in step (1), the final concentration composition of the vitamin components is as follows: vitamin B1 is 4 μM to 8 μM, vitamin B3 is 600 nM to 1000 nM, vitamin B5 is 300 nM to 500 nM, vitamin B6 is 300 nM to 700 nM, vitamin B7 is 2 nM to 6 nM, vitamin B9 is 2 nM to 6 nM, vitamin B12 is 500 pM to 900 pM, and 4-aminobenzoic acid is 40 nM to 80 nM.

[0017] This scheme clarifies the compatibility range of vitamin-based growth factors, supplements essential growth substances that the target group cannot synthesize on its own, and makes up for the lack of trace growth factors in the native environment. It can effectively promote the growth and proliferation of the target group and increase the proportion and abundance of the target group in the culture system.

[0018] The above-mentioned method for culturing aerobic methanogenic microorganisms in mangrove sediments, wherein in step (1), the culture medium is further supplemented with an aerobic methanogenic substrate and an anaerobic methanogenic archaea inhibitor, sodium 2-bromoethanesulfonate; the aerobic methanogenic substrate is selected from any one or more of dimethyl mercaptopropionic acid inner salt, dimethyl sulfide, dimethyl sulfoxide, methylphosphonic acid, and methylamine.

[0019] This scheme can induce the growth and proliferation of target microbial groups with corresponding metabolic capabilities by adding specific substrates; at the same time, by adding specific inhibitors, it can block the metabolic activity of non-target microbial groups, eliminate the interference of non-target microbial metabolic processes, and significantly improve the accuracy of target microbial enrichment.

[0020] The above-mentioned method for culturing aerobic methanogenic microorganisms in mangrove sediments, wherein the final concentration of the aerobic methanogenic substrate is: 50-100 μmol / L of dimethyl mercaptopropionic acid inner salt, 50-100 μmol / L of dimethyl sulfide, 50-100 μmol / L of dimethyl sulfoxide, 50 μmol / L-1 mmol / L of methylphosphonic acid, and 0.5 mmol / L-2 mmol / L of methylamine; and the final concentration of sodium 2-bromoethanesulfonate is 300 μmol / L-800 μmol / L.

[0021] This approach limits the optimal concentration range of substrate and inhibitor, ensuring that the substrate fully meets the metabolic needs of the target microbial community while the inhibitor effectively suppresses the activity of non-target microbial communities, without negatively impacting the normal growth of the target community, thus guaranteeing the long-term stable operation of the culture system.

[0022] The above-mentioned method for culturing aerobic methanogenic microorganisms in mangrove sediments, wherein in step (3), the sediment sample is weighed by wet weight and the solid-liquid ratio of the sediment sample to the culture medium is (5g~15g):90mL.

[0023] This method, by limiting the appropriate ratio of sample to culture medium, can construct a uniform and stable culture system that not only fully matches the physicochemical characteristics of the original environment of the sample, providing a suitable growth microenvironment for the target microgroups, but also ensures that the nutrients in the culture medium are in full contact with the target microbial groups in the sample, thereby improving the utilization efficiency of nutrients and the enrichment effect of the target microbial groups.

[0024] The above-mentioned method for cultivating aerobic methanogenic microorganisms in mangrove sediments includes the following steps: In step (4), the volume ratio of oxygen to monofluoromethane in the mixed gas is (8-10):1; the vacuuming and gas injection operations are repeated 3-5 times; and the needle used for gas injection is 0.4mm-0.6mm in diameter.

[0025] This scheme clarifies the key parameter range for constructing the gas environment, which can stably construct and maintain the gas environment required for the culture system, fully meeting the growth requirements of the target group. At the same time, by limiting the operational details, it can effectively avoid gas leakage problems in the culture system, ensure the long-term stability of the gas environment during the culture process, and avoid interference from the external environment on the culture system.

[0026] The above-mentioned method for culturing aerobic methanogenic microorganisms in mangrove sediments, wherein in step (5), the temperature of the constant temperature culture is 25℃~35℃, and the culture process is conducted in the dark.

[0027] This approach limits the suitable culture temperature range to match the temperature of the target microbial community's native environment, providing a suitable temperature environment for the growth and metabolism of the target community and ensuring that the activity of various enzymes within the target community remains stable. The entire process of cultivation in the dark avoids the growth stimulation of algae and microorganisms by light, as well as the negative impact of light on the core metabolic pathways of the target microbial community. At the same time, it prevents the decomposition of substrates within the system caused by light, ensuring the continuous and stable progress of metabolic processes within the culture system.

[0028] The above-mentioned method for culturing aerobic methanogenic microorganisms in mangrove sediments, wherein in step (1), the mother liquors of the macro-elements, micro-elements, and vitamin components are all sterilized by filtration through a 0.20μm to 0.25μm filter membrane; the sterilization method of the basic salt components is high-temperature and high-pressure sterilization at 115℃ to 121℃ for 15min to 30min.

[0029] This scheme clarifies the aseptic treatment methods and key operational details for each component of the culture medium. Through graded sterilization, it avoids the inactivation of heat-sensitive nutrients during sterilization, ensuring the nutritional effectiveness of the culture medium. It also fully guarantees the aseptic environment of the culture system, avoiding interference from other microbial contamination on the enrichment of target groups.

[0030] The present invention has the following beneficial effects: 1. This invention establishes for the first time a method for cultivating aerobic methanogenic microorganisms specifically adapted to the mangrove sediment environment, filling a technological gap in this field. The culture medium system of this invention simulates the ionic composition and concentration of brackish water in mangroves, adapting to the growth environment of microorganisms in mangrove sediments. Compared with existing culture systems derived from marine and lake sources, it is more suitable for the growth and enrichment of aerobic methanogenic microorganisms from mangroves.

[0031] 2. The method of the present invention has strong compatibility and can support a variety of methyl compounds such as dimethyl mercaptopropionic acid inner salt, dimethyl sulfide, dimethyl sulfoxide, methylphosphonic acid, and methylamine as methanogenic functional substrates. It can selectively enrich aerobic methanogenic microorganisms with different substrate preferences, thus expanding the screening and discovery range of aerobic methanogenic microorganisms.

[0032] 3. By adding sodium 2-bromoethanesulfonate, this invention can effectively inhibit the metabolic activity of anaerobic methanogenic archaea, eliminating interference from the classical anaerobic methanogenesis process; and by adding monofluoromethane to inhibit the aerobic oxidation of methane, it ensures that the methane detected in the culture system comes from the aerobic methanogenesis process, thus guaranteeing the accuracy of the enrichment target.

[0033] 4. The method of the present invention is simple to operate, and the system has good stability and repeatability. By constructing a stable aerobic headspace environment through multiple vacuuming and gas injection operations, and combined with constant temperature and dark culture conditions, it can efficiently enrich aerobic methanogenic microorganisms in mangrove sediments, providing reliable technical support for elucidating the process and mechanism of aerobic methanogenesis in mangrove wetlands and for discovering new aerobic methanogenic functional microorganisms. Attached Figure Description

[0034] Figure 1 The image shows the results of methane production detection at site S4, 24.11.08, of the mangrove forest on Qi'ao Island. Figure 2 The image shows the results of methane production detection at site S4, 24.11.11, of the mangrove forest on Qi'ao Island. Figure 3 The image shows the results of methane production detection at site S3, 24.11.17, of the mangrove forest on Qi'ao Island. Figure 4 The image shows the results of methane production detection from the S3 site 24.11.19 mangrove culture on Qi'ao Island. Figure 5 The image shows the results of methane production detection at site S4, 24.11.15, of the mangrove forest on Qi'ao Island. Figure 6 The image shows the results of methane production detection from the S4 site 24.11.19 mangrove culture on Qi'ao Island. Figure 7 The image shows the results of methane production detection at site S4, 24.11.20, of the mangrove forest on Qi'ao Island. Figure 8 The graph shows the results of methane production detection at site S4 (25.01.17) of the mangrove forest on Qi'ao Island during long-term cultivation. Figure 9 Figure showing the results of methane production and dissolved oxygen detection at site S4 (25.03.21) of the mangrove forest on Qi'ao Island during long-term cultivation; Figure 10 A comparison of methane production from mangrove sediment substrates from different regions after 20 days of culture; Figure 11 Figure showing the results of methanogenesis and dissolved oxygen detection at site S3 (25.04.11) of the mangrove forest on Qi'ao Island; Figure 12 Figure showing the results of methanogenesis and dissolved oxygen detection at site S3 (25.03.21) of the mangrove forest on Qi'ao Island; Figure 13 A flowchart for a method of culturing aerobic methanogenic microorganisms in mangrove sediments. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0036] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual images. They should not be construed as limiting the scope of this application. To better illustrate the embodiments of the present invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual dimensions of the product. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0037] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "inner," and "outer" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present application. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0038] In the description of this invention, unless otherwise explicitly specified and limited, the term "connection" or similar designation indicating a connection between components should be interpreted broadly. For example, it can refer to a fixed connection, a detachable connection, or an integral part; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can refer to the internal communication between two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0039] Reference Figure 1-13 The present invention will be further described in detail below with reference to specific embodiments and comparative examples. Unless otherwise specified, the reagents, consumables and instruments used in the embodiments and comparative examples can be obtained through conventional commercial channels; unless otherwise specified, the methods used are conventional operating methods in the art.

[0040] Example 1: Method for Cultivating Aerobic Methanogenic Microorganisms in Mangrove Sediments The specific steps of the method for culturing aerobic methanogenic microorganisms in mangrove sediments in this embodiment are as follows: 1. Culture medium preparation The culture medium consists of basic salt components, macro-element components, micro-element components, vitamin components, methanogenic functional substrates, methane aerobic oxidation inhibitors, and methanogenic archaea inhibitors. All water used is ultrapure water.

[0041] (1) Preparation of mother liquor: Prepare concentrated mother liquors of macro elements, micro elements and vitamin components respectively. The macro element mother liquor is concentrated 1000 times, and the micro element and vitamin mother liquors are concentrated 10000 times. All mother liquors are filtered through a 0.22μm filter membrane for sterilization and then stored in a refrigerator at 4℃ away from light for later use.

[0042] (2) Basic salt sterilization: Weigh each component of the basic salt according to the final concentration, add ultrapure water to dissolve, and transfer to a 1L blue cap bottle. Seal the bottle mouth with a black rubber stopper and tighten the red open cap. Before sterilization, use an orange long needle to puncture the rubber stopper to balance the air pressure inside the bottle. Place it in a high-temperature and high-pressure sterilizer at 121℃ for 20 minutes. After sterilization, wait for the temperature of the sterilizer to drop below 90℃, open the cap, and immediately remove the needle to avoid contamination by other bacteria.

[0043] (3) Culture medium preparation: After the sterilized basic salt solution has been completely cooled to room temperature, macro-elements, micro-elements and vitamin stock solutions are added to the final concentration in a sterile laminar flow hood. After shaking well, methanogenic functional substrate and sodium 2-bromoethanesulfonate are added. After shaking well again, the pH of the culture medium is adjusted to 7.0 using sterile NaOH and HCl solution.

[0044] In this embodiment, the final concentrations of each component are as follows: Basic salt composition: NaCl 481mM, MgCl2·6H2O 27mM, CaCl2·2H2O 10mM, KCl 9mM, MgSO4·7H2O 2.8mM; Major elemental composition: (NH4)2SO4 is 400 μM, and NaH2PO4 is 50 μM at pH 7.5; Trace element composition: FeCl3·6H2O is 117 nM, MnCl2·4H2O is 9 nM, ZnSO4·7H2O is 800 pM, CoCl2·6H2O is 500 pM, Na2MoO4·2H2O is 300 pM, Na2SeO3 is 1 nM, and NiCl2·6H2O is 1 nM; Vitamin composition: Vitamin B1 6 μM, Vitamin B3 800 nM, Vitamin B5 425 nM, Vitamin B6 500 nM, Vitamin B7 4 nM, Vitamin B9 4 nM, Vitamin B12 700 pM, 4-aminobenzoic acid 60 nM; Methanogenic functional substrate: methylamine, final concentration 1 mmol / L; Sodium 2-bromoethanesulfonate: final concentration 500 μmol / L.

[0045] 2. Sample pretreatment Take surface sediment samples from the mangrove forests of Qi'ao Island and remove large particles such as gravel, twigs, and leaf fragments from the samples in a sterile laminar flow hood for later use.

[0046] 3. Preparation of mud system In a sterile laminar flow hood, 10g of wet weight of the treated sediment sample was weighed using a 10,000-level balance and placed in a sterile beaker. 90mL of the prepared culture medium was added, and the mixture was stirred thoroughly using a sterile rotor and a magnetic stirrer until the sediment and culture medium were completely mixed to form a uniform, particle-free slurry system.

[0047] 4. Construction of a sealed and aerobic environment for the culture system Transfer the entire well-mixed mud system to a sterilized 150mL anaerobic bottle. The actual water capacity of the anaerobic bottle is 175-178mL. Immediately seal the bottle mouth with a sterilized butyl rubber stopper and tighten the aluminum cap with a capping tool to complete the plastic sealing.

[0048] The sealed anaerobic bottle was placed in an anaerobic preparation apparatus, and the headspace inside the bottle was evacuated until a near-vacuum state was achieved. A mixture of oxygen and monofluoromethane gas at a volume ratio of 9:1 was drawn in using a sterile syringe and injected into the anaerobic bottle through a 0.6mm sterile needle. During the injection process, the injection was stopped once the syringe stopper was depressurized and the pressure inside the bottle returned to ambient pressure. The above evacuation-injection operation was repeated 3 times to ensure that the headspace of the anaerobic bottle was completely filled with the aerobic mixture, thus establishing a stable aerobic culture environment.

[0049] 5. Constant temperature incubation The anaerobic bottles, after the aerobic environment has been established, were placed in a 30°C constant temperature incubator and cultured in complete darkness to enrich aerobic methanogenic microorganisms.

[0050] 6. Methane detection During the cultivation process, 1 mL of headspace gas was periodically extracted from the anaerobic bottle using a gas injection needle. The methane concentration in the headspace was then detected by a gas chromatograph. The gas chromatograph can simultaneously detect three greenhouse gases: methane, carbon dioxide, and nitrous oxide. A methane standard curve was pre-set before detection to ensure the accuracy of the results.

[0051] Example 2: Culture of aerobic methanogenic microorganisms in mangrove sediments with different substrates The operation steps in this embodiment are basically the same as those in Example 1, with the only difference being the selection of the methanogenic functional substrate and the setting of the final concentration, as detailed below: Dimethylmercaptopropionic acid inner salt group: The substrate is dimethylmercaptopropionic acid inner salt, with a final concentration of 100 μmol / L; Dimethyl sulfide group: The substrate is dimethyl sulfide, and the final concentration is 50 μmol / L; Dimethyl sulfoxide group: The substrate is dimethyl sulfoxide, and the final concentration is 50 μmol / L; Methylphosphonic acid group: substrate is methylphosphonic acid, final concentration is 50 μmol / L; Mixed substrate group: The substrates were equal proportions of dimethyl mercaptopropionic acid inner salt, dimethyl sulfide, and methylamine, with a total final concentration of 1 mmol / L.

[0052] The remaining culture medium components, operating procedures, and culture conditions are completely consistent with those in Example 1.

[0053] Example 3: Basic Salt Component Range Lower Limit Adaptation Example The operation steps in this embodiment are basically the same as those in Example 1, the only difference being the final concentration setting of the basic salt components of the culture medium, as follows: Basic salt composition: NaCl 400mM, MgCl2·6H2O 20mM, CaCl2·2H2O 5mM, KCl 5mM, MgSO4·7H2O 2mM; The other macroelements, microelements, vitamin components, substrates, inhibitors, operating procedures, and culture conditions are completely consistent with those in Example 1.

[0054] Example 4: Adaptation Example of the Upper Limit of Basic Salt Component Range The operation steps in this embodiment are basically the same as those in Example 1, the only difference being the final concentration setting of the basic salt components of the culture medium, as follows: Basic salt composition: NaCl 550mM, MgCl2·6H2O 35mM, CaCl2·2H2O 15mM, KCl 15mM, MgSO4·7H2O 4mM; The other macroelements, microelements, vitamin components, substrates, inhibitors, operating procedures, and culture conditions are completely consistent with those in Example 1.

[0055] Example 5: Multi-parameter range adaptation of culture conditions The operation steps in this embodiment are basically the same as those in Embodiment 1, with the difference being the following parameter settings: 1. Culture medium sterilization parameters: The basic salt components were sterilized by high temperature and high pressure at 115℃ for 30 min, and the stock solutions of macro elements, micro elements, and vitamins were sterilized by filtration through a 0.22μm filter membrane; 2. Solid-liquid ratio of mud system: Weigh 15g of wet sediment sample and add 90mL of culture medium, with a solid-liquid ratio of 15g:90mL; 3. Construction of an aerobic environment: The volume ratio of oxygen to monofluoromethane in the mixed gas is 10:1. The vacuuming-injection operation is repeated 5 times, and the injection needle is 0.45mm. 4. Culture temperature: The constant temperature culture temperature is set to 25℃; The remaining culture medium components, substrates, inhibitors, and other operating procedures are completely consistent with those in Example 1.

[0056] Example 6: Inhibitor and Substrate Concentration Range Adaptation Example The operation steps in this embodiment are basically the same as those in Embodiment 1, with the difference being the following parameter settings: 1. Methanogenic functional substrate: final concentration of methylamine 2 mmol / L; 2. The final concentration of sodium 2-bromoethanesulfonate, a methanogenic archaea inhibitor, is 800 μmol / L; 3. Major elemental composition: (NH4)2SO4 is 500 μM, and NaH2PO4 at pH 7.5 is 80 μM; 4. Trace element composition: FeCl3·6H2O is 150 nM, MnCl2·4H2O is 15 nM, ZnSO4·7H2O is 1000 pM, CoCl2·6H2O is 800 pM, Na2MoO4·2H2O is 500 pM, Na2SeO3 is 2 nM, NiCl2·6H2O is 2 nM; 5. Vitamin composition: Vitamin B1 is 8 μM, Vitamin B3 is 1000 nM, Vitamin B5 is 500 nM, Vitamin B6 is 700 nM, Vitamin B7 is 6 nM, Vitamin B9 is 6 nM, Vitamin B12 is 900 pM, and 4-aminobenzoic acid is 80 nM; The remaining operating steps and culture conditions are completely consistent with those in Example 1.

[0057] Example 7: Adaptation of Low Solid-Liquid Ratio and Low-Concentration Substrate The operation steps in this embodiment are basically the same as those in Embodiment 1, with the difference being the following parameter settings: 1. Solid-liquid ratio of mud system: Weigh 5g of wet sediment sample and add 90mL of culture medium, with a solid-liquid ratio of 5g:90mL; 2. Methanogenic functional substrate: final concentration of methylamine 0.5 mmol / L; 3. The final concentration of sodium 2-bromoethanesulfonate, a methanogenic archaea inhibitor, is 300 μmol / L; 4. Construction of an aerobic environment: The volume ratio of oxygen to monofluoromethane in the mixed gas is 8:1. The vacuuming-injection operation is repeated 4 times, and the injection needle is 0.5mm in diameter. 5. Incubation temperature: The constant temperature incubation period is set to 35℃; The remaining culture medium components and other operating steps are completely consistent with those in Example 1.

[0058] Comparative Example 1: Culture of existing marine aerobic methanogenic microorganisms in culture medium The only difference between this comparative example and Example 1 is that the culture medium used is a conventional marine aerobic methanogenic microbial culture medium in the prior art, while the other operation steps, culture conditions, substrates and inhibitors are completely consistent with Example 1.

[0059] The marine aerobic methanogenic medium used in this comparative example consisted of: 300 mM NaCl, 50 mM MgCl2·6H2O, 10 mM CaCl2·2H2O, 10 mM KCl, 1 mM NH4Cl, and 0.5 mM KH2PO4. The trace elements and vitamins were prepared using a modified marine culture medium formula.

[0060] Comparative Example 2: Anaerobic Culture System The only difference between this comparative example and Example 1 is that in the headspace environment construction step, a mixture of nitrogen and carbon dioxide with a volume ratio of 8:2 is used instead of a mixture of oxygen and fluoromethane to construct the anaerobic headspace environment. All other operation steps, culture medium formulation, substrate and inhibitor additions are completely consistent with Example 1.

[0061] Comparative Example 3: Culture system without sodium 2-bromoethanesulfonate The only difference between this comparative example and Example 1 is that sodium 2-bromoethanesulfonate is not added to the culture medium. All other operating steps, culture medium formulation, substrate addition, and culture conditions are completely consistent with Example 1.

[0062] Comparative Example 4: Substrate-free blank culture system The only difference between this comparative example and Example 1 is that no methanogenic functional substrate is added to the culture medium. All other operating steps, culture medium formulation, inhibitor addition, and culture conditions are completely consistent with Example 1.

[0063] Comparative Example 5: Sediment Leachate Culture System The only difference between this comparative example and Example 1 is that in the sample processing and culture system construction steps, 10g of wet sediment sample was added to 90mL of sterile ultrapure water, stirred thoroughly, and allowed to stand for 30min. The supernatant was then used as inoculum and added to 90mL of the culture medium of Example 1. No mud system was constructed. All other operation steps, culture medium formulation, substrate and inhibitor addition, and culture conditions were completely consistent with Example 1.

[0064] Comparative Example 6: Culture system exceeding the basal salt concentration range The only difference between this comparative example and Example 1 is that the final concentrations of the basic salt components in the culture medium are: NaCl 300 mM, MgCl2·6H2O 10 mM, CaCl2·2H2O 2 mM, KCl 2 mM, and MgSO4·7H2O 1 mM, which are lower than the lower limit of the defined concentration range. All other operating steps, other culture medium components, substrates, inhibitors, and culture conditions are completely consistent with those in Example 1.

[0065] Comparative Example 7: Culture system exceeding the culture temperature range The only difference between this comparative example and Example 1 is that the isothermal culture temperature is set to 40°C, which exceeds the upper limit of the temperature range defined in this invention. All other operating steps, culture medium formulation, substrate, and inhibitor addition are completely consistent with Example 1.

[0066] Comparative Example 8: Culture systems exceeding the inhibitor concentration range The only difference between this comparative example and Example 1 is that the final concentration of sodium 2-bromoethanesulfonate was set to 1000 μmol / L, which exceeds the upper limit of the defined concentration range. All other operating steps, culture medium formulation, substrate, and culture conditions are completely consistent with Example 1.

[0067] Experimental Example To verify the effectiveness and stability of the method of the present invention, and to clarify the technical advantages of the present invention compared with the control scheme, as well as the rationality of the parameter range defined by the present invention, parallel comparative experiments were conducted using the above embodiments and comparative examples. The specific experimental process and results are as follows: Experimental Example 1: Comparison of Methanogenic Effects Between Parallel Cultures of the Example and Comparative Examples Experimental materials Sample: Sediment sample from site S4 in the mangrove forest of Qi'ao Island; Experimental groups: Examples 1-7, Comparative Examples 1-8, with 3 biological replicates in each group; Culture and detection methods: All groups were cultured simultaneously, and the culture conditions were set according to the parameters of the corresponding examples and comparative examples. The culture was carried out in complete darkness. The headspace methane concentration was detected on the 7th and 17th days of culture, and the dissolved oxygen content of the system was detected simultaneously.

[0068] Experimental results 1. Test results on day 7 of culture The results of headspace methane concentration and dissolved oxygen content measurements on day 7 of culture for each group are shown in the table below:

[0069] 2. Test results on day 17 of culture. The results of headspace methane concentration and dissolved oxygen content measurements on day 17 of culture for each group are shown in the table below:

[0070] 3. Results Analysis (1) In the entire culture period, the culture medium of the 1 to 7 groups of the present invention maintained a stable aerobic environment, the dissolved oxygen content remained in the oxygen-rich range, and significant methane generation was detected in all of them. This proves that the technical solutions within the full parameter range defined by the present invention can effectively support the growth and metabolism of aerobic methanogenic microorganisms in mangrove sediments and achieve stable enrichment of target microorganisms. Among them, the methane production of the 1 group of the present invention was always the highest, which is the optimal implementation method and is consistent with the results of the original multiple batches of independent experiments.

[0071] (2) Comparative Example 1 uses existing marine culture medium, and its methane production is only about 12% of that of Example 1. This proves that the special culture medium designed for the brackish water environment of mangroves in this invention has a significantly better enrichment effect on aerobic methanogenic microorganisms from mangroves compared with conventional marine culture medium, highlighting the targeted nature of the culture medium formulation of this invention.

[0072] (3) Comparative Example 2 is an anaerobic culture system with no dissolved oxygen and 500 μmol / L sodium 2-bromoethanesulfonate added. Only extremely low levels of methane were detected, proving that the classic anaerobic methanogenic archaea have been completely inhibited. The methane detected in the embodiments of the present invention does not come from the anaerobic methanogenic process, but from the methanogenic metabolism of microorganisms under aerobic conditions, which clarifies the aerobic methanogenic specificity of the system of the present invention.

[0073] (4) Comparative Example 3, without the addition of sodium 2-bromoethanesulfonate, produced significantly more methane than Example 1. Combined with the results of Comparative Example 2, it can be seen that the extra methane came from uninhibited anaerobic methanogenic archaea. This proves that by adding sodium 2-bromoethanesulfonate, the present invention can effectively eliminate the interference of anaerobic methanogenesis, ensure that the enriched microorganisms are the target aerobic methanogenic microorganisms, and guarantee the accuracy of the enrichment target.

[0074] (5) Comparative Example 4 was a blank group without substrate, in which only trace amounts of methane at the background level were detected, proving that the methane generation in the system of the present invention comes from the metabolism of added methyl substrates by microorganisms, further verifying the direct correlation between the methanogenesis process and the addition of substrates.

[0075] (6) Comparative Example 5 was inoculated with sediment leachate without constructing a mud system. Its methanogenic yield was only about 25% of that of Example 1, which proves that the in-situ mud system constructed in this invention is more in line with the natural environment of mangrove sediments and can provide a more suitable microenvironment for aerobic methanogenic microorganisms, significantly improving the enrichment effect.

[0076] (7) The parameters of Comparative Examples 6 to 8 exceeded the range defined by the present invention, and their methanogenic production decreased significantly, only within 10% of that of Example 1. This proves that the parameter range defined by the present invention is a reasonable range for achieving efficient enrichment of aerobic methanogenic microorganisms in mangroves. The schemes outside the range cannot achieve the technical effect of the present invention, further verifying the rationality and necessity of the parameter limitation of the present invention.

[0077] Experiment Example 2: Verification Experiment on the Enrichment of Aerobic Methanogenesis at Different Sites in the Mangrove Forest of Qi'ao Island Experimental materials Sample: Sediment sample from site S3 in the mangrove forest of Qi'ao Island; Experimental groups: Example 1 group, Examples 3-7 groups, Comparative Examples 1-8 groups, with 3 biological replicates in each group; Culture and detection methods: exactly the same as in Experiment 1, headspace methane concentration was detected on day 3 and day 5 of culture.

[0078] Experimental results 1. On day 3 of cultivation, significant methane production was detected at the S3 site in the Example 1 group, with an average concentration of 3200 ppm. The average methane concentration in the Example 3 to 7 groups was above 2500 ppm. The methane production trend was consistent with that of the S4 site. The methane production in the methylamine group was significantly higher than that in other substrate groups. The methane production in the Comparative Examples 1 to 8 groups was significantly lower than that in the Example groups, which was completely consistent with the trend of Experiment 1.

[0079] 2. On day 5 of cultivation, the average methane concentration at site S3 in group 1 of Example 1 increased to 5800 ppm, and the average methane concentration in groups 3 to 7 of Examples 3 all exceeded 4500 ppm, with good repeatability among parallel groups. Only trace amounts of methane were detected in the anaerobic system group of Comparative Example 2, the blank group of Comparative Example 4, and groups 6 to 8, further verifying that the method of the present invention has good adaptability and stability to sediment samples from different sites in mangroves, and that the full parameter range defined by the present invention has stable technical effects.

[0080] Experiment Example 3: Compatibility Verification Experiment of Mangrove Sediments from Different Regions Experimental materials Samples: Sediment samples from different mangrove forests were collected from Beilun River Estuary (BLHK), Maowei Sea (MWH), and Shankou (SK) in Guangxi Province, and Sanya River (SYH) and Dongzhai Port (DZG) in Hainan Province. Experimental groups: Samples from each region were divided into three groups: Example 1 methylamine group, Example 3 basic salt limit group, Example 7 low parameter range group, Example 2 DMSP group, and Comparative Example 1 marine culture medium group. Each group had three biological replicates. Culture and detection methods: After incubation at 30℃ in the dark for 20 days, the headspace methane concentration was detected.

[0081] Experimental results 1. In mangrove sediment samples from all regions, the methanogenesis of the methylamine group in Example 1 was significantly higher than that of the DMSP group in Example 2, which is consistent with the experimental results of mangrove samples from Qi'ao Island, proving that methylamine is a universally dominant substrate for aerobic methanogenesis in mangrove sediments from different regions.

[0082] 2. The methanogenic yield of mangrove sediment samples from all regions, including Examples 1, 3, and 7, was more than 5 times that of the marine culture medium group in Comparative Example 1, demonstrating that the method of the present invention has broad regional applicability and can be adapted to sediment samples from different mangrove distribution areas in my country. The parameter range defined by the present invention can cover the habitat characteristics of mangroves in different regions, breaking through the limitation of existing technologies that are only applicable to marine and lake samples.

[0083] Experiment Example 4: Long-term culture stability verification experiment Experimental materials Sample: Sediment sample from site S4 of the mangrove forest on Qi'ao Island; Experimental groups: Example 1 group, Example 3 group, Example 5 group, and Example 7 group, with 3 biological replicates in each group; Culture and detection methods: According to the parameters of the corresponding groups, the cultures were incubated at 30℃ in the dark for 90 days. The headspace methane concentration and dissolved oxygen content in the culture medium were detected on the 30th, 60th and 90th days of culture.

[0084] Experimental results 1. On day 90 of culture, the dissolved oxygen content of the culture medium in groups 1, 3, 5 and 7 of Example 1 remained above 5.0 mg / L, maintaining a stable aerobic environment and no anaerobic state was observed.

[0085] 2. On day 90 of cultivation, the average methane concentration in group 1 of Example 1 reached 42,600 ppm, group 3 of Example 3 reached 38,900 ppm, group 5 of Example 5 reached 35,700 ppm, and group 7 of Example 7 reached 31,200 ppm. All of these achieved long-term stable enrichment of aerobic methanogenic microorganisms, proving that the schemes within the parameter range defined by this invention can achieve long-term stable cultivation results and meet the needs of functional microbial discovery and process mechanism research.

[0086] In summary, this method, targeting the habitat characteristics of mangrove sediments and the growth and metabolic patterns of aerobic methanogenic microorganisms, achieves stable enrichment and cultivation of aerobic methanogenic microorganisms in mangrove sediments by simulating the in-situ growth environment to construct a culture medium system, directionally inducing target microorganisms, and inhibiting the growth of non-target microorganisms. The overall working principle is as follows: Firstly, the design principle of the culture medium system is adapted. Mangrove coastal wetlands are located in the sea-land interface area, and the pore water in the sediments has the unique ionic composition and osmotic pressure characteristics of brackish water. This method simulates the in-situ ionic environment of mangrove sediments by adjusting the composition of the basic salts in the culture medium, avoiding growth inhibition of microorganisms due to sudden changes in osmotic pressure or unsuitable ionic environment, and ensuring the stable and normal physiological and biochemical reactions within the microorganisms. The macroelements in the culture medium provide the essential basic nutrients for microbial growth and reproduction, meeting the material requirements for cell synthesis and proliferation; microelements serve as cofactors for key enzymes in microbial metabolism, especially enzymes involved in methanogenesis, ensuring the normal operation of metabolic pathways; vitamin components supplement growth factors that microorganisms cannot synthesize themselves, compensating for the deficiency of microbial growth factors in the in-situ sediment environment, and promoting the continuous proliferation of microorganisms. During the preparation of the culture medium, the basic salts with strong thermal stability are sterilized by high temperature and high pressure, and the heat-sensitive trace components are prepared into a stock solution and then filtered through a filter membrane for sterilization. This ensures a sterile environment for the culture system and avoids the inactivation of heat-sensitive nutrients due to high temperature, thus ensuring the nutritional effectiveness of the culture medium.

[0087] Secondly, the construction principle of the in-situ simulated enrichment culture system is explained. First, large particulate impurities are removed from the sediment sample to prevent them from disrupting the system's homogeneity and adversely affecting the microenvironment for microbial growth. The treated sediment is then mixed with the culture medium at a fixed ratio and thoroughly stirred to form a homogeneous mud system. This system can maximally replicate the in-situ growth environment of mangrove sediments, preventing metabolic stagnation or death of microorganisms due to drastic environmental changes. Simultaneously, it allows various nutrients in the culture medium to fully contact with the microorganisms in the sediment, ensuring that nutrients are effectively utilized by the microorganisms and providing a continuous and stable nutrient supply for their growth.

[0088] Thirdly, the principle of constructing a stable aerobic culture environment is employed. Using a sealed anaerobic bottle as the culture container, multiple vacuuming and gas injection operations completely replace the original gas in the headspace inside the bottle, constructing and maintaining a stable aerobic headspace environment long-term. This fully meets the oxygen requirements for the growth of aerobic methanogenic microorganisms while isolating them from external contamination. This ensures the stability of the test results and does not negatively affect the growth and metabolism of the microorganisms. Controlling the needle specifications during gas injection avoids damage to the sealing stopper, ensuring the airtightness of the culture system, preventing leaks during cultivation, and maintaining the long-term stability of the aerobic environment within the system.

[0089] Fourthly, the principle of targeted enrichment and interference exclusion of target microorganisms is employed. Methyl compounds are added to the culture medium as functional substrates. These substances are metabolic substrates specifically utilized by aerobic methanogenic microorganisms, and can directionally induce the growth and proliferation of microorganisms with the corresponding substrate metabolic capabilities. This allows these microorganisms to gradually gain population dominance in the culture system, achieving targeted enrichment of the target microorganisms. Simultaneously, methanogenic archaea growth inhibitors are added to the culture medium to specifically inhibit the metabolic activity of anaerobic methanogenic archaea, blocking the methanogenesis pathway under anaerobic conditions and preventing interference from anaerobic methanogenesis in the culture system. The injected mixed gas contains monofluoromethane, which acts as an inhibitor of aerobic methane oxidation in microorganisms. This inhibits the oxidation of methane produced in the culture system, ensuring that the methane produced comes entirely from microbial metabolism under aerobic conditions, thus ensuring that the enriched microorganisms are the target aerobic methanogenic microorganisms.

[0090] Finally, there's the principle of adapting the culture conditions. A constant-temperature, dark culture environment is employed, with the culture temperature matching the in-situ environmental temperature of the mangrove sediments. This provides a suitable temperature environment for microbial growth and metabolism, ensuring the stable activity of various enzymes within the microorganisms, enabling continuous and stable growth, reproduction, and methanogenesis. The completely dark culture process avoids the inhibition of methanogenesis-related metabolic pathways by light, while also preventing light-induced substrate decomposition, ensuring substrate stability and maintaining the continuous and stable metabolic processes within the culture system.

[0091] The entire method, through the comprehensive adaptation design of nutrient system, growth microenvironment, gas environment, metabolic regulation, and culture conditions, constructs an aerobic methanogenic microbial culture system that simulates the in-situ characteristics of mangrove sediments. This allows aerobic methanogenic microorganisms, which have low abundance in the natural environment, to grow stably, metabolize and produce methanogens, and gradually accumulate in the artificial system, solving the problem that existing technologies cannot effectively cultivate aerobic methanogenic microorganisms in mangrove sediments.

[0092] The above are merely preferred embodiments of the present invention and are not intended to limit the implementation methods and protection scope of the present invention. Those skilled in the art should recognize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for culturing aerobic methanogenic microorganisms in mangrove sediments, characterized in that, Includes the following steps: Step (1) Culture medium preparation: Prepare a culture medium suitable for the brackish water environment of mangroves. The culture medium includes basic salt components, macro-element components, micro-element components and vitamin components. Adjust the pH of the culture medium to neutral. Step (2) Sample pretreatment: Take surface mangrove sediment samples and remove large solid particles from the samples; Step (3) Preparation of mud system: Mix the pretreated sediment sample with the culture medium obtained in step (1) according to the preset solid-liquid ratio and stir until a uniform mud system is formed; Step (4) Construction of aerobic culture system: The mud system is transferred to a culture container and sealed. The headspace of the culture container is replaced with gas to construct and maintain an aerobic environment for culture. Step (5) Enrichment culture: Place the sealed culture container in a constant temperature and light-proof environment for culture to complete the enrichment of aerobic methanogenic microorganisms in mangrove sediments.

2. The method for culturing aerobic methanogenic microorganisms in mangrove sediments according to claim 1, characterized in that: In step (1), the basic salt component is sterilized by high temperature and high pressure. The macro-element component, trace element component, and vitamin component are respectively prepared into sterile mother liquor. After the basic salt solution is cooled to room temperature, an appropriate amount of macro-element component, trace element component, and vitamin component are added, and the pH is adjusted to neutral. In step (2), the large solid impurities are gravel and fragments of branches and leaves; In step (3), the sediment sample is measured by wet weight; In step (4), the culture container is a sterile anaerobic bottle, sealed with a sterile rubber stopper and an aluminum cap; the gas replacement operation is as follows: after evacuating the headspace of the anaerobic bottle, a mixture of oxygen and monofluoromethane is injected, and the evacuation and injection operations are repeated to fill the headspace of the anaerobic bottle with a mixture of oxygen and monofluoromethane and restore the pressure inside the bottle to normal pressure.

3. The method for culturing aerobic methanogenic microorganisms in mangrove sediments according to claim 1 or 2, characterized in that, In step (1), the final concentration composition of the basic salt components is as follows: NaCl is 400mM to 550mM, MgCl2·6H2O is 20mM to 35mM, CaCl2·2H2O is 5mM to 15mM, KCl is 5mM to 15mM, and MgSO4·7H2O is 2mM to 4mM.

4. The method for culturing aerobic methanogenic microorganisms in mangrove sediments according to claim 1 or 2, characterized in that, In step (1), the final concentration composition of the macro-element components is: (NH4)2SO4 is 300μM to 500μM, and NaH2PO4 with pH 7.5 is 30μM to 80μM.

5. The method for culturing aerobic methanogenic microorganisms in mangrove sediments according to claim 1 or 2, characterized in that, In step (1), the final concentration composition of the trace element components is as follows: FeCl3·6H2O is 100nM to 150nM, MnCl2·4H2O is 5nM to 15nM, ZnSO4·7H2O is 500pM to 1000pM, CoCl2·6H2O is 300pM to 800pM, Na2MoO4·2H2O is 200pM to 500pM, Na2SeO3 is 0.5nM to 2nM, and NiCl2·6H2O is 0.5nM to 2nM.

6. The method for culturing aerobic methanogenic microorganisms in mangrove sediments according to claim 1 or 2, characterized in that, In step (1), the final concentration composition of the vitamin components is as follows: vitamin B1 is 4 μM to 8 μM, vitamin B3 is 600 nM to 1000 nM, vitamin B5 is 300 nM to 500 nM, vitamin B6 is 300 nM to 700 nM, vitamin B7 is 2 nM to 6 nM, vitamin B9 is 2 nM to 6 nM, vitamin B12 is 500 pM to 900 pM, and 4-aminobenzoic acid is 40 nM to 80 nM.

7. The method for culturing aerobic methanogenic microorganisms in mangrove sediments according to claim 1 or 2, characterized in that, In step (1), the culture medium is further supplemented with an aerobic methanogenic substrate and an anaerobic methanogenic archaea inhibitor, sodium 2-bromoethanesulfonate; the aerobic methanogenic functional substrate is selected from any one or more of dimethyl mercaptopropionic acid inner salt, dimethyl sulfide, dimethyl sulfoxide, methylphosphonic acid, and methylamine.

8. The method for culturing aerobic methanogenic microorganisms in mangrove sediments according to claim 7, characterized in that, The final concentrations of the aerobic methanogenic substrates are: 50–100 μmol / L for dimethyl mercaptopropionic acid inner salt, 50–100 μmol / L for dimethyl sulfide, 50–100 μmol / L for dimethyl sulfoxide, 50 μmol / L–1 mmol / L for methylphosphonic acid, and 0.5 mmol / L–2 mmol / L for methylamine; the final concentration of sodium 2-bromoethanesulfonate is 300 μmol / L–800 μmol / L.

9. The method for cultivating aerobic methanogenic microorganisms in mangrove sediments according to claim 2, characterized in that, In step (3), the sediment sample is measured by wet weight, and the solid-liquid ratio of the sediment sample to the culture medium is (5g~15g):90mL; In step (4), the volume ratio of oxygen to monofluoromethane in the mixed gas is (8-10):1; the vacuuming and gas injection operations are repeated 3-5 times; the needle used for gas injection is 0.4mm-0.6mm in diameter.

10. The method for cultivating aerobic methanogenic microorganisms in mangrove sediments according to claim 2, characterized in that, In step (5), the temperature for constant temperature culture is 25℃~35℃, and the culture process is conducted in the dark. In step (1), the mother liquors of the macro-elements, micro-elements, and vitamin components are all sterilized by filtration through a 0.20μm to 0.25μm filter membrane; the sterilization method of the basic salt components is high-temperature and high-pressure sterilization at 115℃ to 121℃ for 15min to 30min.

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