Rhodococcus aetherivorans capable of fixing methane and application thereof

Abstract

The present application relates to a kind of methane immobilization Rhodococcus aetherivorans, the Rhodococcus aetherivorans is preserved in China Microorganism Culture Collection Center on November 26, 2025, and the classification name is Rhodococcus aetherivorans M323 with CGMCC NO.36783, and the classification name is Rhodococcus aetherivorans M323.The multifunctional Rhodococcus aetherivorans of the present application has high utilization ability to air, and can produce oil and orange yellow high value pigment.The multifunctional Rhodococcus aetherivorans of the present application can participate in carbon sink and greenhouse gas emission reduction by carbon fixation and methane oxidation, can supplement nitrogen source for ecological system by nitrogen fixation, can synthesize oil and pigment, has good application prospect in greenhouse effect management, environmental remediation, biological resource development and high added value product production and other fields, and has good economic value.

Description

Technical Field

[0001] This invention belongs to the field of carbon conservation, emission reduction and environmental functional microbial applications, and more specifically, relates to a methane-fixing Rhodococcus ethereinus and its applications. Background Technology

[0002] carbon dioxide( ) and methane ( These two gases are the first and second largest greenhouse gases, respectively, and their synergistic emission reduction has become the most cutting-edge approach to achieving the "dual carbon" goal. Methanotrophs are currently the only known biological methane sinks; these bacteria convert methane into carbon through ribulose monophosphate or serine pathways. While it can be converted into biomass, its mainstream research and application have long been limited to the single function of methane oxidation. The strains have a single capability dimension, making it difficult to create high added value while reducing carbon emissions.

[0003] Currently, strains used for biomethane sinks mainly face the following three bottlenecks:

[0004] Single function: Most of the reported type I and type II methanogenic bacteria only have the ability to oxidize methane and lack a complex metabolic pathway that simultaneously fixes carbon, fixes nitrogen, produces oil or synthesizes high-value pigments. This results in poor process economics and makes it difficult to form a closed loop of benefits between carbon emission reduction and biomanufacturing.

[0005] Cultivation difficulties: Traditional enrichment-purification-separation processes are time-consuming and yield low results. Furthermore, most methanogenic bacteria have stringent requirements for oxygen concentration, copper ions, and temperature windows, limiting high-density fermentation and scale-up production.

[0006] Low added value of products: The metabolites of existing strains are mainly single-cell proteins or polyhydroxy fatty acids (PHA), which have limited market premium and cannot cover the costs of methane capture, purification and fermentation, resulting in insufficient motivation for commercialization.

[0007] Therefore, discovering and domesticating a novel strain possessing five core capabilities—carbon fixation, methane oxidation, nitrogen fixation, oil production, and pigment production—can not only... and Simultaneously converting these substances into high-value chemicals, reducing exogenous nitrogen addition through nitrogen fixation, and increasing product premiums through pigment synthesis, is seen by the industry as a disruptive direction that breaks through the single emission reduction model and achieves synergistic resource utilization and high-value-added processing of greenhouse gases. However, there are currently no reports on microbial resources that simultaneously integrate the above five functions, both domestically and internationally, necessitating the development of corresponding strains and supporting application technologies. Summary of the Invention

[0008] During our research, our team screened a highly versatile and multifunctional Rhodococcus ethereinus. This strain can use methane, carbon dioxide, etc. as the sole carbon source for autotrophic growth, fix N2 in the air, and has the functions of pigment and lipid synthesis.

[0009] Based on the above research, the present invention provides a methane-fixing Rhodococcus aetherivorans, which was deposited on November 26, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.36783 and taxonomically named Rhodococcus aetherivorans M323.

[0010] The present invention also provides the application of the above-mentioned Rhodococcus ethereinus in the fixation of methane or carbon dioxide.

[0011] The present invention also provides the application of the above-mentioned Rhodococcus ethereans in nitrogen fixation.

[0012] The present invention also provides the application of the above-mentioned Rhodococcus ethereinus in the production of pigments.

[0013] In one specific implementation, the pigment is an orange-yellow pigment.

[0014] The present invention also provides the application of the above-mentioned Rhodococcus ethereinus in the production of oils and fats.

[0015] The multifunctional ether-eating Rhodococcus of the present invention is effective against airborne pathogens. , and It has high utilization capacity and can produce oils and high-value orange-yellow pigments. Therefore, the multifunctional Rhodococcus ethereans of this invention can participate in carbon sequestration and greenhouse gas emission reduction through carbon fixation and methane oxidation, supplement nitrogen sources for the ecosystem through nitrogen fixation, and synthesize oils and pigments. It has good application prospects in the fields of greenhouse effect control, environmental remediation, biological resource development, and high value-added product production, and has great economic value.

[0016] Microbial Preservation

[0017] The algal strain described in this invention was isolated from rice-growing soil in Jiangsu Province, China. Based on 16S rRNA gene sequencing and morphological identification, this strain belongs to *Rhodococcus aetherivorans*. It was deposited on November 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 36783, and named *Rhodococcus aetherivorans* M323. Attached Figure Description

[0018] Figure 1 This is a colony photograph of strain R. aetherivorans M323.

[0019] Figure 2 For strain R. aetherivorans M323 Growth curves for cultures using only one carbon source.

[0020] Figure 3 For strain R. aetherivorans M323 Results of stable isotope labeling.

[0021] Figure 4 For strain R. aetherivorans M323 Growth curves for cultures using only one carbon source.

[0022] Figure 5 Characterization of nitrogen fixation function of strain R. aetherivorans M323.

[0023] Figure 6 The results of Nile Red staining of strain R. aetherivorans M323 are shown. (a) and (c) are fluorescence micrographs and ordinary optical micrographs of bacterial cells stained with Nile Red, respectively; (b) and (d) are fluorescence micrographs and ordinary optical micrographs of bacterial cells not stained with Nile Red, respectively. Detailed Implementation

[0024] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0025] The culture media used in the experiment are as follows.

[0026] LB agar medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L, pH 7.0.

[0027] NMS agar medium: , , , , , 0.2 g; Fe-EDTA, 0.38 g, Add 1 ml of trace element solution and 15 g / L agar.

[0028] The trace element solution has the following composition (g / L): , , , , EDTA-2Na 0.25 g / L , .

[0029] Ashby medium: mannitol 10.0 g, potassium dihydrogen phosphate (… 0.2 g, magnesium sulfate ( 0.2 g of sodium chloride (NaCl), 0.2 g of calcium carbonate ( 5.0 g, calcium sulfate ( 0.01 g, 15 g agar, 1 L sterile water.

[0030] 1. Obtaining the strain

[0031] Soil samples were taken from Yangzhou, Jiangsu Province. Rice rhizosphere soil samples were randomly collected, passed through a 0.6 mm sieve to remove large particles such as gravel and soil clods, and stored at 4℃.

[0032] Microorganisms were extracted from soil samples using density gradient centrifugation as follows: 1) 2 g of soil was weighed into a centrifuge tube, 5 ml of PBS was added, and 25 μl of Tween-20 was added. The mixture was then stirred to prepare a soil suspension. 2) The soil suspension was slowly added to the upper layer of an 80% iohexol solution without disturbing the lower layer. 3) The mixture was centrifuged at 4℃ and 14000g for 90 min to obtain the layered liquid. 4) The cell layer (intermediate layer) was taken, transferred to a new centrifuge tube, washed, and the soil microorganisms were obtained.

[0033] Based on a liquid-phase single-cell Raman sorting coupled culture chip that can highly maintain cell viability, target cells are efficiently sorted according to Raman spectroscopy, coated onto NMS agar medium, and placed in a vacuum-sealed chamber, ensuring that the methane content in the air inside the chamber is 20%. The cells are then cultured at 30°C until obvious single colony formation is observed, thus completing the acquisition of live functional bacteria based on "in-situ" metabolic functions. Single colonies are picked and activated in LB liquid medium for bacterial identification.

[0034] 2. Identification of strains

[0035] After activation, the strain was grown on LB plates under aerobic conditions at 30°C for 6 days. Figure 1 As shown, the colony is orange-yellow, round in shape, dry and wrinkled on the surface, uniform in texture, and has neat edges; the colony is medium in size, with most having a diameter of about 1-2 mm.

[0036] The strain was identified using molecular biology methods, specifically 16S rDNA gene sequencing. The 16S rDNA gene of the strain was amplified using universal primers 27F and 1492R, and its sequence was obtained as shown in SEQ ID NO: 1. Alignment with known sequences in GenBank revealed that the 16S rDNA gene sequence of this strain showed over 99% similarity to that of *Rhodococcus aetherivorans*. Combined with colony morphology, this confirmed that the strain belongs to *Rhodococcus aetherivorans*.

[0037] The strain was deposited on November 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36783, and named Rhodococcus aetherivorans M323, with the Latin name Rhodococcus aetherivorans.

[0038] 3. Verification of the methane oxidation function of strain M323

[0039] 3.1 Growth of strain M323

[0040] To address the challenge of mass transfer between methane and liquid gases, we designed a system... Aeration culture was performed using gas as the sole carbon source, with a gas ratio of [missing information]. The culture medium used was NMS inorganic salt medium. OD was detected at 6 time points, with 3 samples taken at each point, 1 ml each time.

[0041] Growth curves as follows Figure 2 As shown, under this culture method, strain M323 was in its growth adaptation period from 0 to 4 days of culture, with a cell density of ( The value slowly rises to 0.1, with the logarithmic growth phase lasting 4-13 days. It rose rapidly from 0.03 to 0.2, and then gradually entered a plateau period after 13 days.

[0042] 3.2 Stable isotope labeling

[0043] by Aeration culture was performed using gas as the sole carbon source, with a gas ratio of [missing information]. The carbon source ratio was 30% air and 70% air, and the culture medium used was NMS inorganic salt medium. A control group was set up with the following carbon source: The carbon source for the experimental group was All other conditions remained consistent. After 14 days of labeled culture, bacterial cells were collected by centrifugation, freeze-dried overnight, and then subjected to stable isotope tracing experiments to detect bacterial cell abnormalities. value.

[0044] Isotope labeling results as follows Figure 3 As shown, group The value is -28‰. group The value was -4.8‰, indicating a highly significant difference between the two groups. This demonstrates that strain M323 can utilize methane as a carbon source for metabolism and can achieve biotransformation of labeled carbon.

[0045] 4. Verification of photosynthetic carbon fixation function of strain M323

[0046] Under natural light, Spray-based aeration culture was performed using the sole carbon source, with a gas ratio of [missing information]. The culture medium used was NMS inorganic salt medium. OD was detected at 6 time points, with 3 samples taken at each point, 1 ml each time.

[0047] Growth curves as follows Figure 4 As shown, under this culture method, strain M323 was in its growth adaptation period from 0 to 48 hours of culture, with a cell density of ( The value slowly rises to 0.1, and the logarithmic growth phase lasts for 48-96 hours. It rose rapidly from 0.1 to 0.48, reaching its highest value at 96 hours, after which it entered a plateau period.

[0048] 5. Verification of nitrogen fixation function of strain M323

[0049] Assumption agar, a nitrogen-free medium, was used as the functional verification medium. Strains M323 and the control strain E. coli BL21 were simultaneously inoculated onto Assumption agar, a nitrogen-free medium. Strains M323 were able to grow colonies and fix airborne bacteria. No colonies grew in the control group, such as Figure 5 As shown.

[0050] 6. Verification of oil-producing function of strain M323

[0051] by Aeration culture was performed using gas as the sole carbon source, with a gas ratio of [missing information]. The culture medium used was NMS inorganic salt medium. After growth to the logarithmic growth phase, bacterial cells were harvested and stained with Nile red for observation using a fluorescence microscope. The control group consisted of bacterial cells not stained with Nile red.

[0052] After treating strain M323 with Nile Red staining agent, observation under a fluorescence microscope (excitation wavelength λ=480 nm, emission wavelength λ=550 nm) revealed numerous bright yellow fluorescent dots evenly distributed in the field of view. Figure 6 As shown in (a) and (c), the clear fluorescence signal and low background interference indicate that Nile Red dye has specifically bound to lipid droplets within microbial cells and emitted characteristic fluorescence, reflecting the distribution and accumulation level of lipids within the cells. The control group showed no obvious fluorescence signal in the field of view, as... Figure 6 As shown in (b) and (d), only weak background noise exists, proving that the generation of fluorescence signal in this experimental system depends entirely on the specific binding of Nile Red to intracellular lipid droplets, excluding the interference of sample autofluorescence or non-specific staining.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A methane-fixing Rhodococcus aetherivorans, characterized in that, The *Rhodococcus aetherivorans* was deposited on November 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.36783 and taxonomic name Rhodococcus aetherivorans M323.

2. The use of the Rhodococcus ether-eating as described in claim 1 in the fixation of methane or carbon dioxide.

3. The application of Rhodococcus ether-eating as described in claim 1 in nitrogen fixation.

4. The application of the Rhodococcus ethereinus as described in claim 1 in the production of pigments.

5. The application according to claim 4, characterized in that, The pigment is an orange-yellow pigment.

6. The application of Rhodococcus ethereans as described in claim 1 in the production of oils and fats.

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