Methanophilic strain for producing extracellular polymeric substances and method for producing extracellular polymeric substances by using methanophilic strain

Abstract

The invention discloses a methanophilic strain for producing an extracellular polymeric substance and a method for producing the extracellular polymeric substance by using the methanophilic strain, and belongs to the technical field of industrial biology. The methanophilic strain for producing the extracellular polymeric substances is classified and named as extracellular polymeric substance methanophilic bacteria NG09, can grow by using methane or methanol as a unique carbon source, is preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 14, 2024, and has the preservation number of CGMCC No.30951. The methanophilic strain for producing the extracellular polymeric substances has the advantages that the methanophilic bacteria can be used for producing the extracellular polymeric substances, the methanophilic bacteria can be used for producing the extracellular polymeric substances, the methanophilic bacteria can be used for producing the extracellular polymeric substances, and the methanophilic bacteria can be used for producing the extracellular polymeric substances. When the methanophilic strain for producing the extracellular polymeric substance is used for producing the extracellular polymeric substance, carbon emission reduction is realized through microbial carbon sequestration, and a harmless preparation can be produced by utilizing a non-grain-based one-carbon raw material and is widely applied to the fields of environmental restoration, agricultural production and the like.

Description

Technical Field

[0001] The invention belongs to the field of industrial biotechnology, and specifically relates to a methanophilic strain producing extracellular polymers and a method for producing the extracellular polymers by using the strain. Background Art

[0002] Extracellular polymers are high-molecular polymers released by bacteria, fungi, algae and other microorganisms to the cell surface and the external environment during their growth. The main components are polysaccharides, proteins, and a small amount of nucleic acids and other components. As highly hydrated biomacromolecules released by microorganisms during their growth and metabolism, extracellular polymers have been proven to have the effect of alleviating soil environmental stress, such as enhancing water retention, improving salt tolerance, and resisting antimicrobial compounds. However, commercial extracellular polymer products are currently mainly derived from microbial fermentation processes using various types of grains such as starch as raw materials. Not only are they expensive, but they also cause a situation of "competing with people for food." Although there have been reports on the use of environmental microorganisms to produce extracellular polymers, the existing environmental microbial strains have slow growth rates, low cell densities, and poor tolerance to salt stress, which leads to low efficiency in the production of extracellular polymers. Their application scenarios are limited to arable land and cannot be applied to harsh soil environments such as saline-alkali land.

[0003] In addition to fossil resources such as natural gas and shale gas, methane can also come from associated gas in coal seams and oil fields. In addition, the biogas produced by the anaerobic digestion process of various organic waste treatment plants also contains a large amount of methane. At present, these methane-rich gases are mainly used for combustion to produce heat and electricity, which is a single way of utilization and has low added value. Methanophiles can naturally use methane as the only carbon source for growth and can synthesize various biomacromolecules and platform compounds. Methane biomanufacturing technology using methanophiles as cell factories has begun to show potential. Methanophiles are environmental microorganisms that can adapt to various stress environments. Therefore, there is an urgent need to breed methanophiles that can grow rapidly and synthesize extracellular polymers under salt stress, and to develop new methods for producing extracellular polymers using non-food-based raw materials such as methane. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a methanophilic strain that produces extracellular polymers and a method for producing extracellular polymers using the strain, so as to solve the technical problems of high cost of existing extracellular polymer production methods, slow growth rate of methanophilic strains and poor salt stress tolerance.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect of the present invention, a methanophilic strain producing extracellular polymers is disclosed, which can grow with methane as the sole carbon source and is classified and named as Methanogenum Tuvanii ( Methylotuvimicrobium sanxanigenens)NG09, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is June 14, 2024, and the deposit number is CGMCC No.30951.

[0006] The second aspect of the present invention discloses the use of a methanophilic strain producing extracellular polymers in producing extracellular polymers.

[0007] Preferably, the exopolymer-producing methanotrophic strain utilizes methane-rich gas to produce exopolymers.

[0008] Further preferably, the methane-rich gas is coalbed methane, oilfield gas, natural gas, shale gas, biogas or biomethane.

[0009] Preferably, the tolerance concentration of NaCl of the methanophilic strain producing extracellular polymers is 40-180 g / L.

[0010] The third aspect of the present invention discloses a method for producing extracellular polymers, comprising inoculating the seed liquid of the above-mentioned methanophilic strain producing extracellular polymers into a liquid inorganic salt medium for fermentation culture, while introducing methane-rich gas and air; then taking the supernatant of the fermentation culture liquid for alcohol precipitation to precipitate the extracellular polymers; The formula of each liter of liquid inorganic salt culture medium is: 0.6~1.4 g MgSO4·7H2O, 0.002~0.017 gCaCl2·6H2O, 2~15 g KNO3 and 7~150 g NaCl, and the balance is water.

[0011] Preferably, the method for preparing the seed liquid of the methanophilic strain producing extracellular polymers is as follows: transferring the above-mentioned methanophilic strain producing extracellular polymers to a solid inorganic salt culture medium, culturing at 26±6°C for 3-6 days to obtain a bacterial block; inoculating the bacterial block into a liquid inorganic salt culture medium, and shaking culturing at 29±4°C for 30-72 hours to obtain a seed liquid; Among them, the formula of solid inorganic salt culture medium per liter is: 0.6~1.4 g MgSO4·7H2O, 0.002~0.017 gCaCl2·6H2O, 2~15 g KNO3, 7~150 g NaCl and 5~20 g agar powder, and the rest is water.

[0012] Preferably, the inoculation amount of the seed solution is 7% to 20% of the volume of the liquid inorganic salt culture medium.

[0013] Preferably, the volume ratio of methane-rich gas to air is 1:(1-5).

[0014] Preferably, during the alcohol precipitation process, the amount of anhydrous ethanol added is 1 to 6 times the volume of the supernatant.

[0015] The fourth aspect of the present invention discloses the use of a methanotrophic strain producing extracellular polymers in improving the soil environment. The extracellular polymers are produced by fermentation of the methanotrophic strain, and the soil environment is improved by enhancing the soil's water retention capacity and promoting soil aggregation through the extracellular polymers.

[0016] The fifth aspect of the present invention discloses the use of a methanotrophic strain producing extracellular polymers in soil remediation, wherein the extracellular polymers are obtained by fermentation of the methanotrophic strain, and the soil environment is remediated by adsorbing heavy metals and organic pollutants through the extracellular polymers.

[0017] The sixth aspect of the present invention discloses the use of a methanotrophic strain that produces extracellular polymers in protecting plants. The extracellular polymers produced by fermentation of the methanotrophic strains can prevent salt and antibacterial compounds from infiltrating plant roots, and can also prevent the growth of pathogenic microorganisms, thereby improving the tolerance of plants to stress environments.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a methanophilic strain that produces extracellular polymers, which is classified and named as extracellular polymeric methanogens Tuvanella ( Methylotuvimicrobium sanxanigenens )NG09. The strain can tolerate high-concentration salt stress environment and has strong salt tolerance. Second, it can grow with CH4 as the only carbon source, without starch and other types of food as raw materials. It can resource-utilize CH4 and effectively reduce the production cost of extracellular polymers. Third, it can efficiently synthesize extracellular polymers with high water retention capacity and antibacterial activity. Using this methanophilic strain that produces extracellular polymers, methane can be converted into high-value-added products (extracellular polymers) while achieving carbon fixation and carbon emission reduction. It can use non-food-based one-carbon raw materials to produce harmless agricultural preparations, and is widely used in environmental remediation and agricultural production.

[0019] Collection Instructions The present invention preserves the extracellular polymer-producing methanophilic strain as follows: Deposit date: June 14, 2024; Deposit location: Beijing, China. Institute of Microbiology, Chinese Academy of Sciences, General Microbiology Center (CGMCC), No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit number: CGMCC No.30951. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 For the extracellular polymer Methanogen Tuvanii ( Methylotuvimicrobium sanxanigenens ) Photo of NG09 growing on solid inorganic salt medium with methane as the sole carbon source; Figure 2 For the extracellular polymer Methanogen Tuvanii (Methylotuvimicrobium sanxanigenens ) Photo of NG09 growing in liquid inorganic salt medium with methane as the sole carbon source; Figure 3 For the extracellular polymer Methanogen Tuvanii ( Methylotuvimicrobium sanxanigenens ) Microscope observation results of NG09; Figure 4 For the extracellular polymer Methanogen Tuvanii ( Methylotuvimicrobium sanxanigenens NG09's housekeeping gene rpoD Gel electrophoresis results of Figure 5 For the extracellular polymer Methanogen Tuvanii ( Methylotuvimicrobium sanxanigenens ) Growth curve of NG09 cultured in 3 L bioreactor; Figure 6 For the extracellular polymer Methanogen Tuvanii ( Methylotuvimicrobium sanxanigenens ) Physical picture of the extracellular polymers produced by NG09 (after purification); Figure 7 For the extracellular polymer Methanogen Tuvanii ( Methylotuvimicrobium sanxanigenens )Verification diagram of the water retention effect of extracellular polymers extracted from NG09 fermentation supernatant; Figure 8 For the extracellular polymer Methanogen Tuvanii ( Methylotuvimicrobium sanxanigenens )Verification diagram of the heavy metal adsorption effect of extracellular polymers extracted from NG09 fermentation supernatant; Figure 9 For the extracellular polymer Methanogen Tuvanii ( Methylotuvimicrobium sanxanigenens ) Validation diagram of the anti-plant pathogenic effect of extracellular polymers extracted from NG09 fermentation supernatant; Figure 10 For the extracellular polymer Methanogen Tuvanii ( Methylotuvimicrobium sanxanigenens ) Verification of the effect of extracellular polymers extracted from NG09 fermentation supernatant on promoting tomato seed germination. DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0022] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0023] In this article, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0024] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0025] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0026] In this article, the solid inorganic salt culture medium and liquid inorganic salt culture medium used need to be sterilized at 121°C for 20 min before use.

[0027] The present invention provides a methanophilic strain that produces extracellular polymers, which is classified and named as extracellular polymeric methanogens Tuvanella ( Methylotuvimicrobium sanxanigenens )NG09, which can grow with methane or methanol as the only carbon source, was screened from the paddy field soil in Liuxiu Village, Chang'an District, Xi'an City, Shaanxi Province, my country, and was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit date is June 14, 2024, and the deposit number is CGMCC No.30951.

[0028] The present invention provides a method for producing extracellular polymers using the above-mentioned methanophilic strain that produces extracellular polymers, the steps of which are as follows: 1) The extracellular polymer-producing methanophilic strain ( Methylotuvimicrobium sanxanigenens ) NG09 was transferred to solid inorganic salt medium and cultured at 26±6℃ for 3-6 days to obtain bacterial blocks; Among them, the formula of solid inorganic salt culture medium per liter is: 0.6~1.4 g MgSO4·7H2O, 0.002~0.017 gCaCl2·6H2O, 2~15 g KNO3, 7~150 g NaCl and 5~20 g agar powder, and the rest is water.

[0029] 2) inoculating the bacterial mass obtained in step 1) into a liquid inorganic salt culture medium, and culturing with shaking at 29±4°C for 30-72 hours to obtain a seed solution; The formula of each liter of liquid inorganic salt culture medium is: 0.6~1.4 g MgSO4·7H2O, 0.002~0.017 gCaCl2·6H2O, 2~15 g KNO3 and 7~150 g NaCl, and the balance is water.

[0030] 3) inoculating the seed solution prepared in step 2) into a liquid inorganic salt culture medium at an inoculum amount of 7% to 20% by volume, and fermenting for 48 to 96 hours, while continuously adding methane-rich gas and air to the culture system at the beginning and during the culture process to obtain a fermentation liquid; The methane-rich gas includes but is not limited to coalbed methane, oilfield gas, natural gas, shale gas, biogas and biomethane.

[0031] 4) Take the fermentation supernatant obtained in step 3), add 1 to 6 times the volume of anhydrous ethanol to the fermentation supernatant, precipitate overnight, centrifuge at (8000-12000) × g for 15-33 min, discard the supernatant, dry the sample after precipitation, and obtain extracellular polymers.

[0032] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0033] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, are conventional commercial products, and their specifications are conventional specifications in the art.

[0034] 1. Acquisition of methanophilic strains producing extracellular polymers 1. Sample collection: Some soil was collected from the rice fields of Liuxiu Village, Chang'an District, Xi'an City, Shaanxi Province, transported to the laboratory in cold storage, and stored in a 4°C refrigerator.

[0035] 2. Strain enrichment: Take 14-22 g of the soil collected in step 1 and place it in a triangular shake flask, add 50-70 mL of sterile distilled water and 35-55 glass beads, shake at 30-35°C and 100-150 rpm for 40-60 min, let it stand at room temperature for 20 min, and then centrifuge at low speed to obtain soil extract. Take 5 mL of the supernatant of the soil sample extract and add it to the injection bottle containing liquid inorganic salt culture medium, and seal it with a rubber stopper. Use a syringe to inject 50-60 mL of CH4 into the injection bottle, and enrich and culture it at 26-36°C and 200-250 rpm until the fermentation liquid in the injection bottle becomes turbid. The fermentation liquid is applied to a solid inorganic salt medium, and cultured in an anaerobic tank filled with a mixture of CH4 and air for 3 to 6 days, and monoclonal strains with faster growth and larger diameter are selected and cultured in a liquid inorganic salt medium with the same NaCl concentration to obtain a fermentation liquid of a methanophilic strain enriched in extracellular polymers. The formula of each liter of liquid inorganic salt culture medium is: 0.8 g MgSO4·7H2O, 0.012 g CaCl2·6H2O, 2 g KNO3 and 7 g NaCl, and the balance is water; The formula of solid inorganic salt culture medium per liter is: 0.8 g MgSO4·7H2O, 0.012 g CaCl2·6H2O, 2 g KNO3, 7 g NaCl and 12 g agar powder, and the rest is water.

[0036] 3. Purification culture: Take 10-25 μL of the fermentation liquid of the methanophilic strains enriched for extracellular polymer production, streak it on a solid inorganic salt medium, and culture it in an anaerobic tank for 3-6 days. Select the monoclonal strains with faster growth and larger diameter and transfer them to the liquid inorganic salt medium, and repeat the above operation twice. The final methanophilic culture liquid is used for seed preservation to test its tolerance to salt stress.

[0037] 4. Improve the salt stress tolerance of the obtained methanophilic bacteria: The improvement of salt stress tolerance is conducive to the microbial strains to better cope with the salt stress environment, and can significantly increase the viscosity of the fermentation broth. Taking advantage of the characteristics of adaptive evolution of microorganisms under salt stress conditions, the strategy of liquid culture domestication-solid culture verification was adopted, and the methanophilic strains preserved in step 3 were cultured in the order of gradually increasing NaCl concentrations (concentration gradient of 40, 60, 80, 100, 120 g / L NaCl). The monoclonal methanophilic strains that grew stably and rapidly in the solid inorganic salt medium containing 120 g / L NaCl were inoculated into the liquid inorganic salt medium for expansion and preservation. In the order of gradually increasing NaCl concentrations (the concentration gradient increased by 1 g / L NaCl), the strain was inoculated into the liquid inorganic salt medium containing 140~200 g / L NaCl, cultured in a 96-well plate and its upper limit of salt stress tolerance was verified. The results showed that the methanophilic strain could grow normally in liquid inorganic salt medium containing 153 g / L NaCl, and the highest NaCl tolerance concentration was 176 g / L.

[0038] 5. Identification of strains: The methanophilic bacteria tolerant to high NaCl concentrations obtained in step 4 were cultured in solid inorganic salt medium and liquid inorganic salt medium, respectively, and their phenotypes were observed.

[0039] The anaerobic system of rice fields where the strain grows naturally slowly produces and releases methane, with low oxygen content and soil salt concentration of 0.45%. It has been verified that the methanotrophic strain can grow in a wide range of methane / air ratios from 3:97 to 65:35, is more suitable for environments with lower oxygen levels, and has a higher growth rate in the range of methane / oxygen ratios from 0.8 to 1.2. In addition, Figure 1 As shown in the figure, on the solid inorganic salt medium, the colonies grow for 4 days and are light yellow and round, relatively dry, and can be picked up with an inoculation loop. Figure 2 As shown in the figure, during the fermentation process of the liquid inorganic salt medium, the fermentation liquid is white in the early stage, turns orange in the middle stage, and turns light green in the late stage due to decay. The bacteria are Gram-negative bacteria as determined by Gram staining. Figure 3 As shown in the figure, the bacteria were short rod-shaped and had strong motility under microscope observation. Based on the 16S rRNA test results, the genome of the bacteria was extracted and its housekeeping gene was amplified and verified using standard primers. rpoD .like Figure 4 As shown, the first lane in the electrophoresis is the marker, and the second to sixth lanes are the housekeeping genes amplified from the bacterial genome. rpoD , with a length of 190 bp, located between the two marker bands of 100 and 250 bp. Combined with the above results, it was determined that the methanogens belonged to MethylotuvimicrobiumThe genus is a bacterium named NG09, whose Latin name is Methanogenum Tuvanii ( Methylotuvimicrobium sanxanigenens ), deposited on June 14, 2024 at the Institute of Microbiology, Chinese Academy of Sciences, General Microbiology Center, China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No.30951.

[0040] 2. Production of extracellular polymers using methanophilic strains producing extracellular polymers The extracellular polymer Methanogenomyces cytogenes (Methanogenomyces cytogenes) stored at -80°C Methylotuvimicrobium sanxanigenens ) One inoculation loop of the seed liquid of NG09 was picked, and streaked on a solid inorganic salt culture medium for 5 days. One loop of the bacteria was picked and inoculated into a liquid inorganic salt culture medium. The culture was carried out at 30°C and 200 rpm for 24 h to obtain the seed liquid. The seed liquid was inoculated into a 3-L fermenter containing 2 L of liquid inorganic salt culture medium at a volume fraction of 10%. A mixture of CH4 and air with a volume ratio of 1:3 was continuously introduced to start fermentation.

[0041] The absorbance of the bacterial solution was measured at a wavelength of 600 nm using a visible light spectrophotometer, and the extracellular polymer Methanogenin was plotted. Methylotuvimicrobium sanxanigenens ) Growth curve of NG09 cultured in a 3 L bioreactor.

[0042] Growth curve results are as follows Figure 5 As shown, after 54 hours, the absorbance of the fermentation broth reached 50, indicating that the extracellular polymer Methanogenomyces cytogenes ( Methylotuvimicrobium sanxanigenens NG09 strain fermentation can reach a high cell density in a short time and grow faster. This result proves that the extracellular polymer Methanogen Tuvanella ( Methylotuvimicrobium sanxanigenens )NG09 has good robustness and is expected to achieve large-scale production.

[0043] After 60 h of fermentation, the tank was stopped and the fermentation liquid was harvested. 2.5 times the volume of anhydrous ethanol was added to the fermentation supernatant, precipitated overnight, centrifuged at 9500×g for 25 min, the supernatant was discarded, and the sample was dried after precipitation to obtain extracellular polymers, such as Figure 6 shown.

[0044] Methanogens from Tuvania microbiota Methylotuvimicrobium sanxanigenens ) The fermentation products of NG09 were tested for components. The protein and carbohydrate contents were determined by Kjeldahl method and anthrone sulfuric acid method, respectively. The lipid and polypeptide contents were determined by Soxhlet extraction method and biuret method, respectively. The uronic acid and total phenol contents were determined by carbazole sulfate method and Folin-Ciocalteau method, respectively. The results are shown in Table 1.

[0045] Table 1 ( Methylotuvimicrobium sanxanigenens) NG09 fermentation production of extracellular polymer components test results

[0046] As can be seen from Table 1, carbohydrates and proteins are the extracellular polymers of Methanogenetics Tuvanii ( Methylotuvimicrobium sanxanigenens ) are the main components of the extracellular polymers produced by NG09, accounting for a total of 83.6%.

[0047] 3. Verification of the ability of soil to retain water using extracellular polymers produced by methanophilic strains producing extracellular polymers The water holding capacity of mixed soils was measured to evaluate the extracellular polymeric Methanogens Tuvanii ( Methylotuvimicrobium sanxanigenens ) Water-retention capacity of extracellular polymers obtained from NG09 fermentation.

[0048] Sandy soil from the sandy area in eastern Ningxia was dried to constant weight and placed in a desiccator to cool to room temperature. Methylotuvimicrobium sanxanigenens The extracellular polymers obtained by fermentation of NG09 were uniformly mixed with 65 g of dried sandy soil at a mass percentage of 0.5%, 1%, 2% and 4%, respectively, and loaded into a 50 mL syringe tube, weighed and recorded as m0, deionized water was added dropwise until the soil was completely soaked, and the soil was suspended until no water dripped out, and weighed and recorded as m H , the water holding rate of the soil was calculated according to formula 1. At the same time, the dried sandy soil without the addition of extracellular polymers was used as a control.

[0049] (Formula 1) The results are as follows Figure 7 As shown, only 1% of the extracellular polymer Methanogenomyces cytogenes (Methanogenomyces cytogenes) was added to the sandy soil. Methylotuvimicrobium sanxanigenens The extracellular polymers produced by the NG09 strain had a water holding rate of more than 40% in sandy soil, which was nearly 3.5 times higher than that of the original sandy soil. This result proves that the extracellular polymers of Methanogenetics Tuvanella ( Methylotuvimicrobium sanxanigenens ) The extracellular polymers synthesized by the NG09 strain have a very strong water-retaining effect.

[0050] 4. Verification of the efficacy of extracellular polymers produced by methanophilic strains producing extracellular polymers in adsorbing heavy metals and organic pollutants Prepare simulated heavy metal solutions containing 50 mg / L of copper ions and cadmium ions, take 10 mL and place in a conical flask, add 5 mL of extracellular polymer solutions containing 20, 40, 80, 120 and 160 mg / L, respectively, and then add 15 mL of deionized water. The concentrations of extracellular polymers in each group are 10, 20, 40, 60 and 80 mg / L, respectively. Place the conical flask in a constant temperature shaking shaker and shake for 2 h. After the end, dialyze the mixed solution with a dialysis bag for 12 h. Take 1 mL of the solution outside the dialysis bag and measure the concentrations of copper ions and cadmium ions.

[0051] The results are as follows Figure 8 As shown, the extracellular polymer Methanogenum Tuvanii ( Methylotuvimicrobium sanxanigenens The extracellular polymers produced by NG09 fermentation can efficiently adsorb and remove heavy metals such as copper and cadmium, with the highest removal rates reaching 64.5% and 51.7%, respectively. Therefore, this extracellular polymer is expected to be widely used in the wastewater treatment industry containing multiple heavy metals.

[0052] 5. Verification of the efficacy of extracellular polymers produced by methanophilic strains producing extracellular polymers in inhibiting the growth of harmful microorganisms According to the national standard method GB / T 39101-2020, the inhibition zone method for the determination of antibacterial activity of polypeptides, 1.5% agar powder was added to the commercial potato-glucose liquid medium and sterilized by high pressure. The potato-glucose liquid medium was cooled to 50°C, and the indicator fungus Botrytis cinerea was added under sterile conditions and mixed evenly. The mixture was poured into a sterile culture dish and cooled to prepare the test plate. The extracellular polymer Methanogenomyces cytogenes ( Methylotuvimicrobium sanxanigenens ) The extracellular polymers obtained by NG09 fermentation production were re-dissolved, and the ratio of sterile water to extracellular polymers was 0.625:1, 1.25:1, 2.5:1, 5:1 and 10:1, respectively. 500 μL of the obtained extracellular polymer stock solution of different concentrations was added to the test plate with the Oxford cup pressed lightly, pre-diffused in a 4°C refrigerator for 6 h, and the plate was placed in a 37°C constant temperature incubator. After the inhibition zone was clear, the diameter of the inhibition zone was measured with a vernier caliper.

[0053] The results of inhibition zone measurement in each group are as follows Figure 9 As shown, the results show that the extracellular polymer Methanogenomyces tuvarium ( Methylotuvimicrobium sanxanigenens ) The extracellular polymers produced by NG09 fermentation have a significant inhibitory effect on plant pathogens. The extracellular polymers can be used to prepare plant protection agents, which can be applied regularly during the growth of crops to reduce the incidence of crop diseases and improve agricultural production levels.

[0054] VI. Verification of the efficacy of adding extracellular polymers produced by methanophilic strains to salt-stressed soil environments to promote plant growth According to the national standard GB / T 3543.4-1995, the germination test of crop seeds, 0.2%, 0.5%, 1%, 2% and 5% of extracellular polymers were added and mixed evenly in a culture dish as a germination bed. 100 tomato seeds were evenly placed on the germination bed and incubated in a constant temperature box at 25°C. LED lamps were used to provide stable light. Water was added in time to keep the soil from completely losing water. Seed germination was counted for the first time on the 3rd day and for the last time on the 14th day. The number of days with the most germination in each group was recorded as the peak of germination. The calculation of germination potential and germination index refers to formula 2 and formula 3 respectively.

[0055] (Formula 2) (Formula 3) Germination potential and germination index of different amounts of extracellular polymers added and the control group Figure 10 As shown, the results showed that soil addition of extracellular polymeric Methanogens Tuvanii ( Methylotuvimicrobium sanxanigenens The extracellular polymers (EPs) produced by NG09 fermentation can promote the germination of tomato seeds under the stress environment of drought and saline-alkali soil to varying degrees. The germination potential and germination index of the group with 0.5% EEP added reached 76% and 15.4%, respectively, which were 122.8% and 148.4% higher than those of the control group without EEP added.

[0056] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A methanophilic strain producing extracellular polymers, characterized in that: It can grow with methane as the sole carbon source and is classified as the extracellular polymer Methanogenum Tuvanii ( Methylotuvimicrobium sanxanigenens )NG09, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is June 14, 2024, and the deposit number is CGMCC No.30951.

2. Use of the methanophilic strain producing extracellular polymers according to claim 1 in producing extracellular polymers.

3. The use according to claim 2, characterized in that: Methanotrophic strains that produce extracellular polymers utilize methane-rich gases to produce extracellular polymers.

4. The use according to claim 3, characterized in that: The methane-rich gas is coal-bed methane, oilfield gas, natural gas, shale gas, biogas or biomethane.

5. The use according to claim 2, characterized in that: The tolerance of methanophilic strains producing extracellular polymers to NaCl concentrations ranged from 40 to 180 g / L.

6. A method for producing extracellular polymers, characterized in that The seed liquid of the methanophilic strain producing extracellular polymers according to claim 1 is inoculated into a liquid inorganic salt medium for fermentation culture, and methane-rich gas and air are introduced at the same time; then the supernatant of the fermentation culture liquid is taken for alcohol precipitation to precipitate the extracellular polymers; The formula of each liter of liquid inorganic salt culture medium is: 0.6~1.4 g MgSO4·7H2O, 0.002~0.017 gCaCl2·6H2O, 2~15 g KNO3 and 7~150 g NaCl, and the balance is water.

7. A method for producing extracellular polymers according to claim 6, characterized in that: The method for preparing the seed liquid of the methanophilic strain producing extracellular polymers is as follows: transferring the methanophilic strain producing extracellular polymers according to claim 1 to a solid inorganic salt culture medium, culturing at 26±6°C for 3-6 days to obtain a bacterial block; inoculating the bacterial block into a liquid inorganic salt culture medium, culturing at 29±4°C for 30-72 hours with shaking, to obtain a seed liquid; Among them, the formula of solid inorganic salt culture medium per liter is: 0.6~1.4 g MgSO4·7H2O, 0.002~0.017 gCaCl2·6H2O, 2~15 g KNO3, 7~150 g NaCl and 5~20 g agar powder, and the rest is water.

8. Use of the methanophilic strain producing extracellular polymers according to claim 1 in improving soil environment, characterized in that: Extracellular polymers are produced by fermentation of methanophilic strains, which improve the soil environment by enhancing the soil's water retention capacity and promoting soil aggregation.

9. Use of the methanophilic strain producing extracellular polymers according to claim 1 in soil remediation, characterized in that: Extracellular polymers are produced by fermentation of methanophilic strains, and the soil environment is repaired by adsorbing heavy metals and organic pollutants through the extracellular polymers.

10. Use of the methanophilic strain producing extracellular polymers according to claim 1 in protecting plants, characterized in that: Extracellular polymers produced by fermentation of methanophilic strains can prevent salt and antibacterial compounds from infiltrating plant roots, and can also hinder the growth of pathogenic microorganisms and improve the tolerance of plants to stress environments.

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