Methanotrophic bacteria, gas biodegradation agent prepared from the same and use thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a kind of methanotroph and gas biodegradation agent and application prepared by using same.The present application has screened a rhizosphere methylobacterium L1-M1 which grows rapidly and has high utilization rate of methane, can simultaneously utilize oxygen and nitrate as final electron acceptor to oxidize methane into CO2, and has potential to carry out in-situ treatment of methane in deep coal seam.Based on the rhizosphere methylobacterium L1-M1, the present application further provides a product capable of effectively removing methane in deep coal seam, which can be directly injected into the coal seam to treat methane from source, and can safely and properly treat carbon dioxide generated by biological metabolism, so as to realize source pressure reduction, reduction and safe mining of coal seam gas.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a methanogenic bacterium and a gas biodegrading agent prepared therefrom, and its applications. Background Technology

[0002] Coal mine gas (mainly composed of methane) is a major cause of serious safety accidents such as coal and gas outbursts and gas explosions during coal mining. Currently, mine gas control mainly relies on physical drainage technology of "extraction before mining" and roadway ventilation technology of "production determined by ventilation". However, most coal seams are low-permeability and high-absorption coal seams. Simple physical drainage has problems such as long drainage cycle, low drainage rate and drainage blind zone. Although traditional high-pressure water injection into coal seams can drive away gas and reduce dust to a certain extent, the surface tension of ordinary water is high and it is difficult to penetrate and wet the micropores of coal and rock. Moreover, water can only "physically drive away" gas and cannot fundamentally eliminate methane molecules.

[0003] In recent years, the biodegradation technology that uses methanogens to catalytically oxidize methane into carbon dioxide and water at ambient temperature and pressure has provided a new green and environmentally friendly approach for gas control. However, existing biodegradation solutions mostly focus on end-of-mine treatment, that is, spraying or suspending biological agents in roadways to treat gas in mine return airways. Such existing technologies face insurmountable obstacles in practical engineering applications: (1) Extremely low gas-liquid mass transfer efficiency: The roadway ventilation rate is fast and the air volume is extremely large, resulting in a very short residence time of methane gas in the reaction medium containing the agent, which cannot be effectively dissolved and captured by microorganisms. (2) Low substrate concentration leads to bacterial inactivation: The methane concentration in gas is extremely low (usually between 0.1% and 0.75%), which cannot provide sufficient carbon and energy sources for the growth and reproduction of methanogens, resulting in extremely low biodegradation efficiency and making it difficult to apply on a large scale.

[0004] In order to overcome the shortcomings of end-of-pipe treatment, injecting methanogenic bacteria solution into the unmined coal seam through high pressure via borehole (i.e., "source in-situ treatment") has become a more promising technical direction. The high concentration and pressure of gas in the unmined coal seam can provide sufficient substrate for microorganisms. However, the current conventional in-situ bacterial injection technology still has the following shortcomings: (1) Limitation of the oxygen-free environment in deep coal seams: Most methanogenic bacteria are aerobic microorganisms, while the interior of coal seams 50-100m underground is an absolutely anaerobic or oxygen-deficient environment. After conventional bacterial solution is injected into the coal seam, dissolved oxygen will be quickly depleted, causing methanogenic bacteria to suffocate, become dormant or die, and be unable to continuously degrade gas. (2) Risk of secondary disasters caused by metabolic products: Methanogenic bacteria produce a large amount of carbon dioxide gas through metabolism. If it continues to be generated in the closed coal seam pores without treatment, it will not only cause the local carbon dioxide concentration to exceed the standard, causing the risk of asphyxiation of underground workers, but may also form a new abnormal high-pressure gas zone, which violates the original intention of depressurization and desorption.

[0005] In summary, there is an urgent need for a gas biodegrading agent and its application method specifically for in-situ deep coal seams. This technology needs to address the problem of bacterial inactivation due to oxygen deficiency in deep coal seams, overcome the mass transfer barrier of poor permeability of bacterial solutions in low-permeability coal seams, and safely and properly handle carbon dioxide generated by biological metabolism, thereby achieving source pressure reduction, quantity reduction, and safe mining of coal seam gas. Summary of the Invention

[0006] The purpose of this invention is to provide a methanogenic bacterium, a gas biodegrading agent prepared therefrom, and its applications.

[0007] To achieve the above-mentioned objectives, the technical solution adopted in this invention is: Methylomonas rhizoryzae L1-M1, which was deposited on April 15, 2026, at the China General Microbiological Culture Collection Center (CGMCC), at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.38271.

[0008] Correspondingly, bacterial preparations were prepared using the aforementioned rhizosphere methylmona L1-M1.

[0009] Accordingly, the application of the rhizosphere methylmona L1-M1 or the bacterial preparation in methane degradation.

[0010] Accordingly, a gas biodegrading agent is provided, comprising the rhizosphere methylmona L1-M1 or the bacterial preparation thereof. The gas biodegrading agent further comprises an electron acceptor, activated carbon, and a biosurfactant. Preferably, iron ions and / or copper ions are attached to the activated carbon. More preferably, the gas biodegrading agent further comprises calcium peroxide and glucono-δ-lactone.

[0011] Accordingly, the application of the gas biodegrading agent in degrading methane and treating coal mine gas is preferably carried out by injecting the gas biodegrading agent into the coal seam.

[0012] This invention offers the following advantages: It screened and obtained a rapidly growing rhizosphere methylmona strain, L1-M1, with a high methane utilization rate. This strain can simultaneously utilize oxygen and nitrate as the final electron acceptor to oxidize methane to CO2, demonstrating its potential for in-situ methane removal from deep coal seams. Based on the aforementioned rhizosphere methylmona strain L1-M1, this invention further provides a product capable of effectively removing methane from deep coal seams. The product includes modified activated carbon, which acts as a microcarrier, adsorbing methane from the air and creating a localized high-concentration methane environment. Simultaneously, bacteria attached to the activated carbon surface can directly utilize the adsorbed methane, achieving a dynamic cycle of "adsorption-biodegradation-adsorption site regeneration." Preferably, the activated carbon is loaded with iron and copper ions. Iron ions can form a micro-electrolysis system, increasing electron transfer, while copper ions can activate the pMMO enzyme activity of the methane-oxidizing bacteria, accelerating methane oxidation.

[0013] The biggest challenge in coal seam water injection is that the hydrophobicity of coal makes it difficult for water to penetrate the micropores. The gas biodegrading agent provided by this invention contains rhamnose esters, which not only reduce surface tension and promote bacterial solution penetration, but also greatly increase the solubility of gas in the aqueous phase, enhance gas-liquid-solid mass transfer, and improve methane degradation efficiency.

[0014] The main product of methane oxidation is CO2. Although CO2 is not easily explosive, reducing the risk of coal seam explosion, if it is generated and accumulated in large quantities in the coal seam, local carbon dioxide concentrations may exceed the standard during mining, posing a risk of worker asphyxiation. Furthermore, deep coal seams are absolutely anaerobic environments. If methanogenic bacteria are injected into the coal seam under high pressure and then sealed, the bacterial solution will quickly deplete the trace amounts of dissolved oxygen, then "suffocate" and go dormant or die, unable to continuously consume methane. To address this problem, this invention adds calcium peroxide to the product. Calcium peroxide slowly releases O2, generating Ca(OH)2. The generated O2 can be used by methanogenic bacteria to oxidize CH4 and produce CO2. CO2 is converted to carbonate ions in the alkaline environment of Ca(OH)2 and reacts with Ca... 2+ Calcite (CaCO3) is precipitated. This provides the oxygen needed by the methanogenic bacteria and converts the gaseous CO2 products into solid calcite, thus solving the problem of localized excessive carbon dioxide concentration.

[0015] Calcium peroxide has certain bactericidal and bacteriostatic effects, which can reduce the activity of methanogenic bacteria and even lead to their death. Therefore, this invention further adds GDL to the product to neutralize the excessive alkalinity generated by calcium peroxide, successfully transforming the "bactericide" into an "oxygen-supplying agent," thus achieving the coexistence of microorganisms and peroxide. Attached Figure Description

[0016] Figure 1 This is a colony morphology diagram of rhizosphere methylmonas L1-M1. Detailed Implementation

[0017] This invention provides a novel methanogenic bacterium, *Methylomonasrhizoryzae* L1-M1, which was deposited on April 15, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO. 38271. The 16S rDNA sequence of strain L1-M1 is shown in SEQ ID NO: 1.

[0018] The rhizosphere methylmona L1-M1 can simultaneously utilize oxygen and nitrate as the final electron acceptor to oxidize methane into CO2.

[0019] Based on the aforementioned methanogenic bacteria, the present invention also provides a gas biodegrading agent. The gas biodegrading agent comprises: the rhizosphere methylmona L1-M1, modified activated carbon (preferably with attached Fe and Cu as adsorbents and carriers), rhamnose lipolipids (biosurfactant), glycerol (moisturizing and carbon source), calcium peroxide micropowder, glucono-δ-lactone (GDL), and sodium nitrate (electron acceptor). Preferably, by weight, the methane dust remover comprises: 100 parts of bacterial solution (effective viable bacteria count ≥ 10). 8 CFU / mL), rhamnolipin 0.05–0.2 parts, activated carbon or modified activated carbon 0.1–0.5 parts, glycerol 0.5–2.0 parts, calcium peroxide micro powder 0.25–2.5 parts, glucono-δ-lactone 0.05–0.1 parts.

[0020] The present invention also provides a method for preparing the gas biodegradable agent, comprising the following steps:

[0021] 1. Culture of bacterial strain: Culture Methylmonas L1-M1 to the logarithmic growth phase to obtain seed culture.

[0022] 2. Matrix compounding: Take the seed liquid and add sodium nitrate, rhamnolipid, activated carbon or modified activated carbon, glycerol, calcium peroxide micro powder, and gluconate-δ-lactone in sequence. Stir magnetically for more than 30 minutes to ensure complete dissolution and uniform dispersion, thus obtaining the gas biodegrading agent. In practice, it is preferable to first mix the seed liquid with the other substances except for the calcium peroxide micro powder, and then add the calcium peroxide micro powder when it is ready for use.

[0023] Modified activated carbon with attached Fe or Cu can be prepared using methods known in the art, and one optional preparation method is provided:

[0024] (1) Pretreatment of activated carbon: Wash the activated carbon with water to remove dust and ash; Acidification and oxidation: Soak the activated carbon in a 60°C water bath for 4 hours with 10% dilute nitric acid at a solid-liquid ratio of 1:10 (g / mL) so that the nitric acid can oxidize the carbon surface to generate -COOH and -OH, thereby improving hydrophilicity and metal anchoring points; Then wash repeatedly with pure water until the filtrate is neutral, and dry in an oven at 105°C for 12 hours.

[0025] (2) Preparation of Fe / Cu mixed impregnation solution: Prepare a mixed solution of 0.2 mol / L FeCl3 and 0.04-0.05 mol / L CuCl2.

[0026] (3) Constant temperature impregnation loading: according to the ratio of 1g activated carbon to 10mL metal mixed solution, the pretreated activated carbon is put into the mixed solution and impregnated in a shaker at 50℃ and 160r / min for 22-24h, so that the metal ions are fixed in the carbon skeleton by complexation of surface functional groups and adsorption in pores.

[0027] (4) Wash gently with deionized water 2 to 3 times to remove free floating ions on the surface, and then dry at low temperature of 80°C for 12 hours to obtain modified activated carbon with attached Fe and Cu.

[0028] The present invention also provides an optional method of using the gas biodegrading agent, comprising the following steps:

[0029] 1. Drilling Construction: Use a ZDY drilling rig to drill hole C1, insert a pressure gauge, and observe the initial gas pressure for 7 days until the pressure stabilizes. Drill test holes J1, J2, and J3 in sequence, sealing each hole tightly immediately after completion, and installing pressure gauges and sampling valves. Finally, drill injection hole Z1.

[0030] 2. High-pressure sealing: The Z1 hole adopts a pressurized grouting method of "plugging both ends and injecting in the middle", and the sealing length is not less than 12m. A clean water pressure resistance test must be carried out to ensure that there is no leakage under a pressure of 8MPa.

[0031] 3. Water fracturing: Connect the high-pressure pump unit to borehole Z1. Inject clean water at a low flow rate of 50 L / min. After the pressure stabilizes, gradually increase the flow rate to 100 L / min. Observe the pressure curve. When the pressure drops significantly (peak fracturing pressure, expected to be 8-12 MPa), it indicates that new fractures have formed in the coal seam. Continue injecting water for 30 minutes to expand the fractures.

[0032] 4. Gas biodegradable agent injection: Mix the gas biodegradable agent (all components except calcium peroxide) and water at a ratio of 1:10 (v / v), then add calcium peroxide and inject the solution. Switch the water source to the gas biodegradable agent tank.

[0033] Injection pressure: Controlled at 4–8 MPa (below the rupture pressure to prevent ineffective loss of the gas biodegradable agent). Injection flow rate: 80–120 L / min. Total injection volume: 200 m³ 3 (Continuous or intermittent injection). Recording: Record pressure and flow changes throughout the process, once every 30 minutes.

[0034] 5. Well-closing reaction: After injection is completed, immediately close the Z1 orifice valve. Enter the "well-closing" stage, which lasts for 7 days.

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the data obtained are all average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0036] The culture media and reagents used in the examples are as follows:

[0037] NMS medium (g / L): MgSO4•7 H2O 0.75, KNO3 0.5, CaCl2•2H2O 0.2, Na2HPO4•12 H2O 0.25, KH2PO4 0.15, agar 13.0, trace elements 1 ml / L, pH=6.8.

[0038] Trace elements (g / L): ZnSO4•7H2O 0.43, CoCl2•6H2O 0.1, Na2MoO4 0.06, H3BO3 0.1.

[0039] Example 1: Screening and Identification of Strains

[0040] 1. Screening

[0041] The enriched and domesticated bacterial population was serially diluted (10⁻⁶). -1 ~10 -7 After sterilizing and cooling the NMS solid medium, pour it into plates. Once solidified, use a pipette to transfer 200 μL of each graded dilution into the plates, spread evenly, and then place them in a gas incubator with a CH4:O2 volume ratio of 1:1. Incubate at 30°C in the dark for 7 days. The target bacterial strain was characterized by pale red colonies (methanogenic bacteria), transparent, raised, glossy, and moist. These characteristics were used for screening, and preliminary judgment was made through microscopic examination. A methanogenic bacterium, L1-M1, was obtained through screening.

[0042] 2. Purification, preservation and identification of bacterial strains

[0043] The methanogenic bacteria L1-M1 were inoculated onto a new NMS solid medium. After the colonies grew, the best-growing colonies were selected and streaked onto plates. The culture was subcultured 3-4 times, and the purified strain was stored in NMS slant medium at 4°C.

[0044] Long-term preservation of methanogenic bacteria L1-M1 was achieved using the glycerol cryopreservation method. The desired strain was inoculated onto NMS medium and cultured under suitable conditions until the logarithmic growth phase. Five portions of this culture were taken and mixed with two portions of the culture preservation solution. Approximately 0.2 mL was dispensed into culture preservation tubes, tightly capped, and placed at -20°C. The next day, one tube was thawed and inoculated onto NMS medium, ensuring good growth and no contamination. The remaining tubes could then be stored long-term.

[0045] The colony morphology of methanogenic bacteria L1-M1 is as follows: Figure 1 As shown, it is pale red, transparent, raised, glossy, and moist. Methanogenic bacteria L1-M1 are Gram-negative bacteria that can utilize methane as their sole carbon source and nitrates and ammonium salts as nitrogen sources. The optimal growth temperature is 25–35°C, and the temperature they can tolerate does not exceed 40°C. The optimal growth pH is 7–8, and the pH they can tolerate does not exceed 9.

[0046] Methanogenic bacteria L1-M1 were inoculated into NMS liquid medium and cultured at 30℃ and 180 rpm for 48 h to obtain fermentation broth. 1 mL of the fermentation broth was used for 16S rRNA amplification and sequencing. The 16S rRNA of L1-M1 was amplified using universal primers 27F and 1492R. The amplified product was subjected to agarose gel electrophoresis, and a bright band appeared at approximately 1400 bp. The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, yielding a 1421 bp sequence. The sequence was submitted for BLAST alignment, and the results showed a 99.86% per.identity with *Methylomonas rhizoryzae* (MF521886.1), identifying strain L1-M1 as *Methylomonas rhizoryzae*.

[0047] 3. Strain L1-M1 utilizes oxygen and nitrates to oxidize methane.

[0048] Add 100 mL of NMS medium (sodium nitrate-free) to each of four 250 mL anaerobic bottles. Randomly select two bottles and add 1 g / L sodium nitrate to each as the anoxic group. Inoculate each of the four bottles with 5% (v / v) L1-M1 seed culture. In the anoxic group, purge with N2 for 5 min to replace the air. Afterward, stopper the bottles with rubber stoppers and inject 30 mL of methane into each group. The headspace gas ratio in the aerobic group is CH4:O2 = 1:1 (volume ratio). The anoxic group contains only CH4 and no oxygen, and the medium contains nitrate. Place each group in an incubator and incubate with shaking at 30 °C. Measure the methane content after 1 hour.

[0049] The results showed that the methane content in both groups was 0, indicating that strain L1-M1 can efficiently oxidize methane under both aerobic and hypoxic conditions.

[0050] Example 2: Preparation and Effect Demonstration of Gas Biodegradable Agent

[0051] 1. Preparation of Gas Biodegradable Agent

[0052] The strain L1-M1 was inoculated into NMS medium and cultured at 30°C until the logarithmic growth phase to obtain the seed culture.

[0053] Take 100 mL of bacterial culture and add 1 part sodium nitrate; 0.1 part rhamnolipid; 0.5 part Cu-modified activated carbon; 2.0 part glycerol; 0.25 parts calcium peroxide powder; and 0.08 parts glucono-δ-lactone in sequence. Stir magnetically for 30 minutes to ensure complete dissolution and uniform dispersion, thus preparing the gas biodegrading agent. This serves as the experimental group. In practice, to reduce the long-term toxicity of calcium peroxide powder to microorganisms, it is preferable to prepare and use it immediately. The bacterial culture and other substances can be mixed and prepared in advance, and then the calcium peroxide powder can be added and mixed thoroughly before use.

[0054] Meanwhile, control group 1 was set up with all other conditions the same except that modified activated carbon was not added; control group 2 was set up with all other conditions the same except that rhamnolipid was not added; control group 3 was set up with all other conditions the same except that calcium peroxide powder was not added; and control group 4 was set up with all other conditions the same except that glucono-δ-lactone was not added.

[0055] 250 mL sealed serum bottles were used. 50 mL of the prepared gas biodegrading agent (for either the experimental or control group) was added to each bottle, which was then sealed and 50 mL of methane was injected. Each bottle was placed in a shaker at 30°C (150 rpm), and the headspace methane concentration was detected by gas chromatography after 1 hour. Three replicates were set for each group, and the average value was taken. The results are shown in Table 1.

[0056] Table 1. Remaining methane content in the bottles after 1 hour of treatment for each group.

[0057]

[0058] The results showed that the methane removal efficiency of the experimental groups was significantly improved compared to the control groups. This indicates that activated carbon had a significant adsorption and enrichment effect, rhamnolipid had a significant solubilizing effect, and calcium peroxide powder could slowly release oxygen in situ, solving the problem of limited oxygen transfer inside the low-permeability coating in coal mines. This kept the methane-oxidizing bacteria in a highly active aerobic state, thus synergistically improving the methane removal efficiency. Furthermore, its slow release characteristic had no toxic effect on the bacteria. Compared to the control group without glucono-δ-lactone, the experimental groups showed the most significant improvement in methane removal efficiency, indicating that even without the protection of glucono-δ-lactone, the strong alkali released by the calcium peroxide powder after oxygen release had a very significant inhibitory effect on bacteria, highlighting the importance of the protective effect of glucono-δ-lactone.

[0059] Example 3: Preparation and Effect Demonstration of Gas Biodegradable Agent

[0060] One ton of coal was compressed into dense coal blocks, placed in a sealed container, and 6 m³ of methane was slowly introduced as a pilot test.

[0061] 1. Drilling Construction: Use a small drilling rig to drill hole C1, insert a pressure gauge, and observe the initial gas pressure for 7 days until it stabilizes. The initial gas pressure is 1.2 MPa. Drill test holes J1, J2, and J3 in sequence. Immediately after completion, seal the holes tightly and install pressure gauges and sampling valves. Finally, drill injection hole Z1.

[0062] 2. High-pressure sealing: The Z1 hole adopts a pressurized grouting method of "plugging both ends and injecting in the middle", and the sealing length is not less than 12m. A clean water pressure resistance test must be carried out to ensure that there is no leakage under a pressure of 8MPa.

[0063] 3. Water fracturing: Connect the high-pressure pump unit to borehole Z1. Inject clean water at a low flow rate of 50 L / min. After the pressure stabilizes, gradually increase the flow rate to 100 L / min. Observe the pressure curve. When the pressure drops significantly (peak fracturing pressure, expected to be 8-12 MPa), it indicates that new fractures have formed in the coal seam. Continue injecting water for 30 minutes to expand the fractures.

[0064] 4. Bacterial Solution Injection: Prepare the gas biodegrading agent according to the method of the experimental group in Example 2. First, prepare the bacterial solution, then add other substances sequentially, and dilute with water at a ratio of 1:10 (v / v). Calcium peroxide is solid and is added last to obtain the degradation agent. Switch the water source to the tank containing the degradation agent. Injection pressure: Controlled at 4–8 MPa (below the rupture pressure to prevent ineffective loss of the degradation agent). Injection flow rate: 80–120 L / min. Total injection volume: 200 m³ 3 (Continuous or intermittent injection). Recording: Record pressure and flow changes throughout the process, once every 30 minutes.

[0065] 5. Well-closing reaction: After injection is completed, immediately close the Z1 orifice valve. Enter the "well-closing" stage, which lasts for 7 days.

[0066] Seven days later, the gas pressure was measured and found to be less than 0.75 MPa, and the methane content was less than 4 m³. This indicates that the gas biodegradation agent has a significant effect in eliminating gas and can meet the extraction standards.

[0067] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A gas biodegradable agent, characterized in that: The gas biodegrading agent includes rhizosphere methylmona ( Methylomonas rhizoryzae L1-M1, Cu-modified activated carbon, rhamnolipid, glycerol, calcium peroxide powder, gluconate-δ-lactone and sodium nitrate; The rhizosphere methylmona L1-M1 was deposited at the China General Microbiological Culture Collection Center on April 15, 2026, with accession number CGMCC NO.38271.

2. The application of the gas biodegrading agent according to claim 1 in the degradation of methane.

3. The application of the gas biodegrading agent according to claim 1 in the treatment of coal mine gas.

4. The application according to claim 3, characterized in that: The gas biodegrading agent is injected into the coal seam.

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