Strains with methane oxidation ability and their applications

Abstract

The present invention belongs to the field of microorganisms, and discloses a methane-oxidizing strain and its application. The strain G-5 with methane-oxidizing function provided by the present invention is Bacillus velezensis Bacillus velezensis , G-5 can grow with methane as the sole carbon source and oxidize methane. When cultured in NMS medium with methane gas introduced, the strain grows well, the effect of methane oxidation is remarkable, and the methane emission reduction rate reaches 32.1%.

Description

Technical Field

[0001] The present invention relates to strains with methane oxidation ability and their applications, belonging to the field of microbial technology. Background Art

[0002] With the rapid development of the livestock industry in China, the greenhouse gases generated during the breeding process have been increasing year by year. Methane gas is one of the main components of greenhouse gases. Methane in livestock and poultry breeding mainly comes from the manure excreted by animals and their own intestinal metabolism. However, there is currently no good way to deal with this methane, and it is generally directly discharged into the air. Therefore, achieving carbon emission reduction in the livestock and poultry breeding process is an inevitable way to achieve the healthy development of the livestock industry. Therefore, it is necessary to explore a biological means to achieve green and pollution-free treatment of livestock manure management. Therefore, microorganisms with high-efficiency methane oxidation ability are screened and inoculated into the breeding bedding to reduce methane emissions from the source of livestock and poultry breeding, providing a theoretical basis and technical support for green and low-carbon emission reduction in the livestock industry. Summary of the Invention

[0003] The problem to be solved by the present invention is to achieve carbon emission reduction in the livestock breeding process, and provide a strain with high-efficiency methane oxidation ability and its application. The strain uses methane as a carbon source, and adding the strain to the breeding bedding can achieve carbon emission reduction in the process of livestock manure management.

[0004] To solve the above technical problems, the technical solutions provided by the present invention are as follows:

[0005] A strain with methane oxidation ability was deposited with the China General Microbiological Culture Collection Center on January 22, 2024, with the deposit number CGMCC NO. 29723, and the taxonomic name is Bacillus velezensis Bacillus velezensis , and the deposit location is Beijing.

[0006] The present invention also provides a microbial agent with methane oxidation ability, and the microbial agent contains the above-mentioned strain with methane oxidation ability.

[0007] The present invention also provides a preparation method of the above-mentioned microbial agent with methane oxidation ability, and the preparation method includes the following steps:

[0008] Inoculate the above-mentioned strain into the culture medium at an inoculation amount of 3%, and culture it in a constant temperature shaking incubator at a temperature of 28°C and a rotation speed of 180 r / min for 48 h to prepare a seed solution; mix the cultured seed solution with rice hulls.

[0009] Furthermore, the preparation method of the culture medium is: mix peptone, yeast powder, sodium chloride and distilled water, adjust the pH to neutral with sodium hydroxide, and sterilize it by high pressure.

[0010] The present invention also provides the application of the above-mentioned strain with methane oxidation ability in methane emission reduction during livestock and poultry breeding.

[0011] Furthermore, the above-mentioned microbial agent with methane oxidation ability is added to the breeding bedding.

[0012] Furthermore, the microbial agent is added once every 4 to 6 days.

[0013] Furthermore, the thickness of the breeding bedding is 35 - 45 cm; the water content is 35 - 45%; the turning and throwing depth is 20 - 30 cm.

[0014] Furthermore, the temperature condition is 20 - 50 °C.

[0015] Furthermore, the pH of the breeding bedding is 5 - 9.

[0016] The present invention provides the application of a strain with the ability to oxidize methane, including the following steps:

[0017] (1) Inoculate the G-5 bacterial liquid into the culture medium at an inoculation amount of 3%, and culture it in a constant temperature shaking incubator at a temperature of 28 °C and a rotation speed of 180 r / min for 48 h to prepare a seed liquid;

[0018] (2) Mix the cultured seed liquid with rice hulls.

[0019] Furthermore, the culture solution is: 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride, 1000 ml of distilled water, adjust the pH to neutral with sodium hydroxide, and autoclave at 121 °C for 15 min.

[0020] (3) The composite microbial agent prepared according to the above method is inoculated into the breeding bedding, and by adjusting the thickness, water content, and turning and throwing depth of the breeding bedding, methane generated in livestock and poultry manure management is reduced.

[0021] Furthermore, the composite microbial agent is added to the breeding bedding at an inoculation amount of 0.1% by mass fraction.

[0022] Furthermore, the thickness of the breeding bedding is 40 cm; the water content is 41%; the turning and throwing depth is 24 cm. Beneficial effects

[0023] The strain screened by the present invention can be suitable for growth in the breeding bedding, has high methane oxidation activity. In livestock breeding under the same environmental conditions, the methane emission flux of the experimental group added with the G-5 microbial agent is significantly lower than that of the control group, and the methane emission reduction amount reaches 32.1% compared with the control group. The microbial agent prepared by the present invention has significant carbon emission reduction ability. Description of the drawings

[0024] Figure 1is the Bacillus velezensis described in the present invention Bacillus velezensis Bacterial cell photograph of strain G5;

[0025] Figure 2 is the Bacillus velezensis described in the present invention Bacillus velezensis Phylogenetic tree of strain G5;

[0026] Figure 3 shows the effects on methane emissions under different treatments in litter breeding;

[0027] Figure 4 shows the effects on cumulative methane emissions under different treatments in litter breeding. Specific embodiments

[0028] The present invention will be further described below. The following examples are only used to more clearly illustrate the technical solution of the present invention and should not be used to limit the protection scope of the present invention.

[0029] Samples were collected from a farm, sludge, and a paddy field respectively. The collected samples were subjected to primary screening including continuous enrichment, separation and purification, and rescreening of the strains including determination of methane oxidation ability and adaptability screening under different temperature, pH, and oligotrophic concentration conditions to obtain the methane highly efficient oxidation strain G-5. Its bacterial cell photograph and phylogenetic tree are as shown in Figure 1 and Figure 2 shown.

[0030] Biological material sample preservation:

[0031] G-5 has been preserved in the China General Microbiological Culture Collection Center, preservation address: China General Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, preservation number is CGMCC NO. 29723, and the preservation date is January 22, 2024.

[0032] Bacillus velezensis Bacillus velezensis 16S rRNA sequence of strain G5:

[0033]

[0034] Example 1: Determination of the methane oxidation ability of G-5 and cultivation under different growth conditions

[0035] The specific method is as follows:

[0036] (1) Inoculate 1 mL of the bacterial suspension of G-5 strain into a serum bottle containing 40 mL of NMS liquid medium, install an aluminum-plastic cap to seal the bottle mouth, insert a syringe into the aluminum-plastic cap to extract the air in the bottle and inject an equal amount of methane gas, and cultivate with methane as the sole carbon source. Then apply a small amount of white glue to the pinhole part of the aluminum-plastic cap to prevent air leakage. Use a gas chromatograph to detect the initial concentration of methane in the bottle as J 0 , place the serum bottle in a constant temperature shaker at 30 °C and 170 r / min for 5 d, and then detect the remaining concentration of methane J 1 in the serum bottle, and set up a blank group for comparison.

[0037] (2) Inoculate the bacterial suspension of G-5 strain into 30 mL of sterilized LB liquid medium at an inoculation amount of 1.0%, set the temperatures to 10, 20, 30, 40, and 50 °C respectively, set 3 parallels for each temperature condition, place it in a shaker at 170 r / min, cultivate for 48 h, measure the absorbance value of the bacterial liquid, and study the effect of different temperatures on the growth of the strain.

[0038] (3) Inoculate the bacterial suspension of G-5 strain into 30 mL of sterilized LB liquid medium at an inoculation amount of 1.0%, set the pH values to 4, 5, 6, 7, 8, and 9 respectively, set 3 parallels for each pH condition, place it in a shaker at 170 r / min, cultivate for 48 h, measure the absorbance value of the bacterial liquid, and study the effect of different pH values on the growth of the strain.

[0039] (4) After inoculating the bacterial suspension of G-5 strain into the LB liquid medium diluted to 500 times, 1000 times, 2000 times, and 5000 times respectively at an inoculation amount of 1.0%, set 3 parallels for each culture condition, place it in a shaker at 170 r / min, cultivate for 48 h, measure the absorbance value of the bacterial liquid, and study the effect of different oligotrophy on the growth of the strain.

[0040] The results are shown in Table 1:

[0041] Table 1

[0042]

[0043] According to Table 1, G-5 can survive normally under the condition of oligotrophy of 1000 times. After 5 d of cultivation, the methane oxidation rate reaches 41.89%.

[0044] Example 2: Effect verification test of G-5

[0045] (1) The verification test was carried out in the cowshed. 25 cows with basically the same growth traits and periods were introduced into the experimental group; in the control group, the same number of cows with similar growth conditions as the experimental group were introduced, and the verification experiment lasted for 15 days.

[0046] (2) Two different treatments were designed in the experiment as follows. Experimental group: The G-5 bacterial agent was sprinkled into the cow dung return bedding at an inoculation amount of 0.1% by mass fraction (the bacterial content of the bacterial agent was 2 billion / g, and it was added once every 5 days); Control group: The same bedding was laid in the breeding pen. The bedding thickness was 40 cm; the moisture content was 41%; the turning and throwing depth was 24 cm. The bacterial agent was a mixture of strains and rice hulls, and the bacterial content reached 20×10 8 CFU / g.

[0047] Bedding preparation method: The main components of the bedding are various organic matters such as straw powder, wood chips, mushroom residues, manure residues, and biogas residues. After mixing evenly, the bulk density ≤ 0.45 and the water content ≤ 60%. High-temperature composting fermentation was carried out for 7 - 10 days, and the water content dropped below 45%. After meeting the harmless treatment requirements, it can be spread out and cooled for standby.

[0048] The preparation method of the bacterial agent includes:

[0049] 1) The G-5 bacterial liquid was inoculated into the medium at an inoculation amount of 3%, and cultured in a constant temperature shaking incubator at a temperature of 28°C and a rotation speed of 180 r / min for 48 h to prepare a seed liquid; The medium was: 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride, 1000 ml of distilled water, adjusted to neutral pH with sodium hydroxide, and autoclaved at 121°C for 15 min.

[0050] 2) The cultured seed liquid was mixed with rice hulls.

[0051] 3) The static chamber-gas chromatograph method was used to collect and analyze methane generated in the cowshed. Gas was collected on the 0th, 3rd, 6th, 9th, 12th, and 15th days of the experiment. The collection time each time was from 7:00 to 10:00 in the morning. Gas at the 0th, 5th, 10th, and 15th minutes was collected at each point. After being filled into a 0.5 L aluminum foil gas sampling bag, it was measured using a gas chromatograph.

[0052] The results are as Figure 3 shown. The methane emission fluxes of the experimental group and the control group generally showed a fluctuating change trend over time, and this change was closely related to the environmental temperature. In the early stage of the experiment, the sharp drop in environmental temperature led to a significant decrease in the methane emission fluxes of both groups within 0 d - 6 d. The control group dropped to the minimum value of 0.7 mg·m -2 ·h -1 on the 6th day, and the experimental group dropped to 0.5 mg·m on the 6th day-2 ·h -1 。During the later stage of the experiment, the increase in ambient temperature led to a gradual increase in the methane emission fluxes of both groups. In the control group, the methane emission flux had increased to 1.2 mg·m -2 ·h -1 by the 12th day, while in the experimental group, it fluctuated slightly around 0.5 mg·m -2 ·h -1 and continued to rise until the end of the experiment. During the experiment, the initial methane emission fluxes of the control group and the experimental group were relatively close. As time went by, the greenhouse gas emission reduction effect of the experimental group with the addition of G-5 inoculant gradually became apparent. The methane emission flux of the experimental group was significantly lower than that of the control group. On the 12th day, the difference in methane emission fluxes between the two treatments reached the maximum of 0.7 mg·m -2 ·h -1 or so.

[0053] As can be seen from Figure 4 , the cumulative methane amount in the control group reached 2333 mg·m -2 by the 15th day, while the cumulative methane emission of the experimental group was 1584 mg·m -2 . Compared with the control group, the methane emission reduction was 32.1%. The comparison results between the two treatments showed that under the same environmental conditions for breeding, the methane emission flux of the experimental group with the addition of G-5 inoculant was significantly lower than that of the control group.

[0054] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A strain having methane oxidation ability, characterized in that: The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 22, 2024, with the deposit number CGMCC NO. 29723, and the classification name was Bacillus Velezii ( Bacillus velezensis ), the storage location is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A bacterial agent having methane oxidation ability, characterized in that: The bacterial agent contains the bacterial strain having methane oxidation ability according to claim 1.

3. The method for preparing a bacterial agent having methane oxidation ability according to claim 2, characterized in that: The preparation method comprises the following steps: The strain described in claim 1 is inoculated into a culture medium at a 3% inoculation rate, and cultured in a constant temperature shaking incubator at a temperature of 28° C. and a rotation speed of 180 r / min for 48 hours to prepare a seed solution; the cultured seed solution is mixed with rice husks.

4. The method for preparing a bacterial agent having methane oxidation ability according to claim 3, characterized in that: The preparation method of the culture medium is as follows: peptone, yeast powder, sodium chloride and distilled water are mixed, pH is adjusted to neutral with sodium hydroxide, and high pressure sterilization is performed.

5. Use of the strain with methane oxidation ability according to claim 1 in reducing methane emissions in livestock and poultry breeding processes.

6. The use according to claim 5, characterized in that: The bacterial agent with methane oxidation ability as claimed in claim 2 is added to the breeding litter.

7. The use according to claim 6, characterized in that: The bacterial agent is added once every 4 to 6 days.

8. The use according to claim 6, characterized in that The thickness of the breeding litter is 35~45 cm; the moisture content is 35~45%; and the turning depth is 20~30 cm.

9. The use according to claim 6, characterized in that: The temperature conditions are 20~50℃.

10. The use according to claim 6, characterized in that: The pH of the breeding litter is 5~9.

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