Bacterial strain for reducing N2O emission of soil with massive application of biogas slurry and application method of bacterial strain

Abstract

The invention belongs to the technical field of environmental microorganisms, and relates to a bacterial strain for reducing N2O emission of soil with a large amount of applied biogas slurry and an application method thereof, the bacterial strain is Pseudomonas sp.K33, and is preserved in China Center for Type Culture Collection, the preservation address is number 299 of eight road, Wuchang District, Wuhan City, Hubei Province, the preservation number is CCTCC M 20242032, the preservation date is September 23, 2024, and the 16S rRNA gene sequence of the bacterial strain is shown as SEQ ID NO.1. According to the invention, the biogas residue is used as a matrix to enrich and separate the efficient N2O reducing strain Pseudomonas sp.K33 in the biogas residue, and the strain is applied to farmland soil to effectively promote the conversion of NO3-NO2-N2O-N2, shorten the conversion period of excess NO3 <-> and NO2 <-> in the soil, reduce the emission of N2O in the farmland soil after a large amount of biogas slurry is applied for a long time, and reduce the emission of greenhouse gases.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental microorganisms, and particularly relates to a strain for reducing N2O emissions in soils with large amounts of biogas slurry and an application method thereof. Background Art

[0002] Nitrous oxide (N2O) is a powerful greenhouse gas with a warming effect 300 times that of CO2, which has a significant impact on global warming. About 42% of the N2O emitted into the atmosphere each year comes from farmland soil. In agricultural activities, long-term excessive fertilization has become a major factor in increasing soil N2O emissions while meeting human demand for crop yields. In addition, after fertilization, nitrates will accumulate in farmland soil below the surface, posing a threat to groundwater.

[0003] Deep application of biogas slurry is one of the effective methods to reduce nitrate accumulation in farmland soil, which can reduce nitrate leaching by about 30.97%, but at the same time it will cause the problem of increased N2O emissions and cause greenhouse gas pollution. At present, the method of alleviating N2O emissions from farmland soil is mostly to inoculate microbial agents in addition to using nitrification inhibitors. The application of microbial amendments is an environmentally friendly and economical method for improving farmland soil. The inoculated microorganisms are diverse, such as Bradyrhizobium and Azospirillum. These strains reduce N2O to N2 through nitrous oxide reductase (N2OR), thereby reducing N2O emissions in farmland soil. Although inoculation of N2O reducing bacteria can reduce N2O emissions from farmland soil, the large-scale production and use of N2O reducing bacteria are costly, and the survival time of the strains in farmland soil is unknown. A Norwegian team used biogas slurry to selectively enrich and isolate fast-growing N2O-reducing bacteria, Cloacibacterium, and applied the bacterial agent to farmland soil, and found that it can indeed reduce N2O emissions in the soil.

[0004] However, these studies only focus on the emission of soil N2O, and do not pay attention to the accumulation of nitrates in the soil. The denitrification process in the soil is mostly carried out step by step by denitrifying microorganisms with different functions. This will cause a large amount of N2O emissions from the denitrification process, and the conversion time of nitrates and nitrites in the soil is long, which may also affect the growth of plants. Summary of the invention

[0005] In order to solve the problems existing in the above-mentioned prior art, the present invention provides a strain and application method for reducing the N2O emission of soil in which a large amount of biogas slurry is applied. In the present invention, biogas residue is used as a matrix to enrich and separate the efficient N2O reducing strain Pseudomonas sp.K33, and the strain is applied to farmland soil to effectively promote NO3- —NO2 - —N2O—N2 conversion, shortening the excess NO3 - and NO2 - The conversion cycle can be shortened to reduce N2O emissions from farmland soils that have been subjected to long-term and large-scale application of biogas slurry, thereby reducing greenhouse gas emissions.

[0006] The specific technical solution adopted by the present invention is:

[0007] A strain for reducing N2O emissions from soil subjected to large-scale application of biogas slurry. The strain is Pseudomonas sp.K33, which is deposited in the China Center for Type Culture Collection at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC M 20242032 and a deposit date of September 23, 2024. The 16S rRNA gene sequence is shown in SEQ ID NO.1.

[0008] A method for applying a strain to reduce N2O emissions from soils that are heavily slurried, comprising the following steps:

[0009] The Pseudomonas sp.K33 strain is prepared into a Pseudomonas sp.K33 bacterial agent, and then the Pseudomonas sp.K33 bacterial agent is inoculated into biogas slurry, and then the biogas slurry is applied to farmland soil.

[0010] Furthermore, the preparation method of the Pseudomonas sp.K33 bacterial agent specifically comprises the following steps:

[0011] S1, taking the feces digestate after anaerobic fermentation and placing it in a culture bottle A and sealing the culture bottle A, then replacing the air in the culture bottle A with helium, and then adding N2O to the culture bottle A for enrichment culture;

[0012] S2. After the enrichment culture is completed, the enrichment culture sample and sterile water are added to a sterile centrifuge tube, and cultured at 26-30°C for 25-35 minutes. After standing, the supernatant is taken for gradient dilution and spread on the TSA medium solid respectively, and then the TSA medium solid coated with the supernatant is placed in an air bag filled with N2O concentration of 4-6% and sealed for purification to obtain the strain Pseudomonas sp.K33;

[0013] S3, take another culture bottle B, add TSA medium liquid to culture bottle B, inoculate OD 600 The Pseudomonas sp.K33 strain was 0.4-0.6, the culture bottle B was sealed and cultured at 26-30℃. 600When the pH value is 0.8-1, the culture is terminated to obtain the bacterial solution of Pseudomonas sp.K33;

[0014] S4. Centrifuge the Pseudomonas sp. K33 bacterial solution in culture bottle B at 3800-4200 r / min, discard the supernatant, and then resuspend the bacterial cells in sterile 1× phosphate buffer to obtain a Pseudomonas sp. K33 bacterial agent.

[0015] Furthermore, 0.22-26 mL N2O is added to each gram of fecal sludge in the culture bottle A.

[0016] Furthermore, in step S2, the solid content of the TSA culture medium per liter includes: 12-18 g of tryptone, 4-6 g of soy peptone, 4-6 g of sodium chloride, and 12-18 g of agar.

[0017] Furthermore, in step S3, each liter of the TSA culture medium liquid includes: 12-18 g of tryptone, 4-6 g of soy peptone, and 4-6 g of sodium chloride.

[0018] Furthermore, the mass ratio of the Pseudomonas sp.K33 bacterial agent to the biogas slurry is 1-2:1000, and then the biogas slurry is applied to the farmland soil at a dosage of 4-5 cubic meters per mu.

[0019] The beneficial effects of the present invention are:

[0020] 1. In the present invention, the highly efficient N2O reducing strain Pseudomonassp.K33 is enriched and separated from the biogas residue as a matrix, and the strain is applied to the farmland soil to effectively promote NO3 - —NO2 - —N2O—N2 conversion, shortening the excess NO3 - and NO2 - The conversion cycle can be shortened to reduce N2O emissions from farmland soils that have been subjected to long-term and large-scale application of biogas slurry, thereby reducing greenhouse gas emissions.

[0021] 2. In the present invention, biogas residue is used as a matrix to enrich and separate the highly efficient N2O reducing strain Pseudomonassp. K33, which can grow stably in biogas residue and has a great advantage over the strain isolated from soil.

[0022] 3. The N2O reduction capacity of Pseudomonas sp. K33 in the present invention is 9.53±0.87mmol / d / L, which can reduce the daily N2O emission by up to 78% in the field, thereby reducing greenhouse gas pollution caused by farmland soil.

[0023] 4. After the Pseudomonas sp.K33 in the present invention is combined with biogas slurry and applied to a 15 cm soil layer, it can promote nitrate and nitrite to produce N2O and reduce it to N2. Compared with the mechanism of action of the Clausii strain, it has an additional mechanism of promoting nitrate to produce N2O, which can reduce the cumulative amount of nitrate nitrogen in the 15-30 cm soil layer by 13% and reduce the risk of nitrate leaching.

[0024] 5. The strains in the present invention can quickly convert excess nitrate into N2O and reduce it to N2 for emission without affecting the sufficient nitrate in the farmland soil to provide nutrients for the plants, thereby reducing greenhouse gas pollution while alleviating nitrate leaching, providing technical support for the research and development of environmentally friendly bio-biogas fertilizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the accumulation of N2O in the process of enrichment and cultivation of biogas residue;

[0026] Figure 2 The reduction rate of N2O in the process of enrichment culture of biogas residue;

[0027] Figure 3 is the denitrification capacity of Pseudomonas sp.K33 strain;

[0028] Figure 4 is the N2O reduction capacity of Pseudomonas sp.K33 strain;

[0029] Figure 5 A static box device for collecting N2O emissions in the field;

[0030] Figure 6 The daily N2O emissions from deep application of biogas slurry (ZN) and deep application of biogas slurry plus bacterial solution (ZJ) in the field;

[0031] Figure 7 It is the cumulative N2O emission from deep application of biogas slurry (ZN) and deep application of biogas slurry plus bacterial solution (ZJ) in the field;

[0032] Figure 8 Phylogenetic tree of Pseudomonas sp.K33 strain based on 16S rDNA sequence analysis. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0034] A strain for reducing N2O emissions from soils with large amounts of biogas slurry, the strain being Pseudomonas sp.K33, deposited in China Center for Type Culture Collection, with a deposit address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC M 20242032, and a deposit date of September 23, 2024, and a 16S rRNA gene sequence as shown in SEQ ID NO.1, specifically as follows:

[0035]

[0036]

[0037] The 16S rRNA gene sequence of K33 was compared with the NCBI (National Center for Biotechnology Information) database. The results showed that the 16S rRNA gene sequence of strain K33 had a sequence similarity of 99.78% with that of Pseudomonas sp. strain MN889018.1. The phylogenetic tree was constructed using MEGA7.1 ( Figure 8 ), forming a stable evolutionary branch with the sequence of Pseudomonas sp. strain MN889018.1. 1. Specific embodiments

[0039] Example 1

[0040] Preparation of Pseudomonas sp.K33 inoculant

[0041] Sample source: Cow dung residue after anaerobic fermentation;

[0042] TSA medium solids: tryptone 15g / L, soy peptone 5g / L, sodium chloride 5g / L, agar 15g / L, adjust pH to 7.2-7.4, and sterilize by high pressure steam at 121℃ for 30min;

[0043] TSA medium liquid: tryptone 15g / L, soy peptone 5g / L, sodium chloride 5g / L, adjust the pH to 7.2-7.4, and sterilize by high pressure steam at 121°C for 30min.

[0044] S1. Take 20g of biogas residue and put it into a 120mL serum bottle A. Seal the serum bottle A with a rubber stopper and an aluminum cap. Use an automatic vacuum pump and helium filling device to replace the air in the serum bottle A with helium. Repeat 5 times. After the last helium filling, use a syringe to inject 5mL of N2O into the serum bottle A, and balance the pressure in the bottle with a 5mL syringe filled with 2mL of deionized water.

[0045] During this period, the gas generated in the bottle was detected by the GC-Robot automatic sampling and measurement system. The gas in serum bottle A was collected once a day to detect the N2O content in the bottle. After the N2O gas in the bottle decreased to about 1%, 5mL N2O was added to serum bottle A. The culture was continued for 15 days until the N2O reduction rate stabilized. It was found that the N2O reduction rate reached a peak on the 6th to 8th day, and the reduction rate was as high as 13.51μmol vail -1 d -1 , see Figure 1 and Figure 2 ;

[0046] S2. After the enrichment culture is completed, 0.5 g of the sample on the 7th day of enrichment culture is taken and mixed with 20 mL of sterile water in a 50 mL sterile centrifuge tube, and placed in a constant temperature shaker at 28°C and 180 r / min for 30 min. After standing for 1 hour, an appropriate amount of supernatant is taken for gradient dilution, and spread on the solid TSA culture medium, respectively, and placed in an air bag filled with 5% N2O and sealed, and cultured at 28°C until colonies appear. The texture of the colonies and the concave-convex properties of the edges, the morphology and color of the hyphae in the base and the aerial hyphae are observed, and 5 different single colonies are selected and inoculated into the TSA culture medium liquid, and placed in a constant temperature shaker at 28°C and 180 r / min for culturing until the culture medium becomes turbid; the above turbid bacterial liquid is respectively plated and streaked on the solid TSA culture medium until a single colony appears, and a single colony is repeatedly picked and streaked for 3 times to obtain the strain Pseudomonassp.K33;

[0047] S3. Add 2.5L TSA medium into a 3L conical flask and inoculate 2mL OD 600 The strain was 0.5 and placed in a constant temperature shaker at 28°C and 180 r / min for culture. 600 When the value reaches 1, the culture is terminated to obtain the bacterial solution of Pseudomonas sp.K33;

[0048] S4. Centrifuge the Pseudomonas sp. K33 bacterial solution at 4000 r / min, discard the supernatant culture medium, and resuspend the bacterial cells in 1 L of sterile 1x phosphate buffer (PBS) to obtain a Pseudomonas sp. K33 bacterial agent.

[0049] 2. Performance Testing

[0050] 1. Detection of denitrification ability of Pseudomonas sp.K33 strain

[0051] Take serum bottle B, add 50 mL of liquid TSA medium (final concentration of KNO3 is 2 mM) into serum bottle B, put it into a rotor (10×3 mm), and measure the OD of the target strain. 600Value, according to OD 600 The inoculum volume of Pseudomonassp. K33 was calculated by using the value × inoculum volume (mL) = 0.5. The serum bottle B was sealed with a rubber stopper and an aluminum cap. The air in the serum bottle B was then replaced with helium using an automatic vacuum pumping and helium filling device. This was repeated four times. After the last helium filling, the pressure in the bottle was balanced with a 5 mL syringe containing 2 mL of 75% ethanol. The concentrations of O2, N2O, and N2 in the serum bottle B were measured every 2 hours at 700 rpm and 28°C using the GC-Robot automatic sampling and measurement system. For details, see Figure 3 .

[0052] According to calculations, Pseudomonas sp.K33 has completely converted NO3 - Reducing to N2, the maximum denitrification rate during this period was 1.39±0.14g NO3 - -N / d / L.

[0053] 2. Detection of N2O reduction ability of Pseudomonas sp.K33 strain

[0054] Take serum bottle C, add 50 mL of liquid TSA medium to serum bottle C, put it into a rotor (10×3 mm), and use a UV spectrophotometer to measure the absorbance value of the target bacteria at a wavelength of 600 nm (i.e. OD 600 Inoculate 1 mL of OD in culture flask C. 600 The value of Pseudomonas sp.K33 bacterial liquid was 0.5, and the serum bottle C was sealed with a rubber stopper and an aluminum cap. Then, the air in the serum bottle C was replaced with helium using an automatic vacuum and helium filling device. This was repeated 4 times. After the last helium filling, 1 mL of N2O was injected into the serum bottle C using a syringe, and the pressure in the bottle was balanced with a 5 mL syringe containing 2 mL of 75% ethanol. The bottle was cultured at a speed of 700 rpm to test the N2O reduction ability of the strain. During the culture period, the gas generated in the bottle was transferred to the gas chromatograph for measurement through an automatic sampling device and a peristaltic pump. The gas in the serum bottle C was collected every 2 hours to detect the reduction of N2O. For details, see Figure 4 .

[0055] According to calculations, Pseudomonas sp. K33 reduced all N2O to N2 after 4 hours, during which the N2O reduction rate could reach a maximum of 9.53±0.87mmol / d / L.

[0056] 3. Application of Pseudomonas sp.K33

[0057] Six equal areas of similar soil conditions were divided in the farmland soil of the experimental site of the integrated farming and breeding area and marked as I, II, III, IV, V, and VI. They were arranged in three rows and two columns. The area of ​​a single area was 10m×10m=100m 2 , corn is planted at 6,000 plants per mu.

[0058] Example: In areas I, IV, and V, a toothed trencher was used to deeply apply biogas slurry (ZJ) containing bacterial agents. The amount of biogas slurry applied was 4 cubic meters per mu. In the example, the mass ratio of bacterial agents to biogas slurry was 1:1000, and a total of about 200 kg N hm was applied. -2 ;

[0059] Comparative example: In areas II, III, and VI, biogas slurry (ZN) was applied using a toothed trencher at a rate of 4 m3 / mu, or about 200 kg N hm -2 ;

[0060] N2O emission data collection: The static box method was used to detect the field N2O emission flux in each area on the 1st, 3rd, 5th, 7th, 9th, 11th and 13th day after fertilization. The static box device used was a closed chamber made of PVC (60cm long, 20cm wide, and 40cm high). Before collecting gas samples, the static box was placed vertically in a 5cm deep groove on the base and sealed with water. Figure 5 As shown. A fan is set on the top of the static box to fully mix the gas in the box. Gas is taken at intervals of 0, 15, 30, and 45 minutes through a three-way valve. Before sampling, the plants in the base are trimmed to prevent the release of greenhouse gases by plant respiration. Gas samples are collected between 8:00 and 11:00 in the morning because this period is generally considered to represent the average daily flux emissions. After the gas sampling is completed, the soil surface temperature and 5cm soil temperature are recorded. A gas chromatograph is used to determine the N2O concentration in the collected samples.

[0061] Gas emission flux calculation formula:

[0062] F=dc / dt×M / V0×H×273 / (273+T)×P / P0

[0063] Where: F is the emission flux (mg·m -2 ·h -1 );Dc / dt time change of target gas concentration in the sampling box;M is the relative molecular mass of N2O (44g / mol), V0 is the volume of ideal gas (22.41g / mol); H is the height of the sampling box (m); T is the average temperature in the sampling box (℃); P is the air pressure at the sampling point (Pa), and P0 is the standard atmospheric pressure (Pa).

[0064] The interpolation and numerical integration methods were used to estimate the total N2O emissions within 13 days after fertilization.

[0065] Data analysis: The daily and cumulative N2O emissions were statistically analyzed using the R language T-test (P<0.05 indicated a significant difference). The results showed that the daily N2O emissions of the ZJ treatment were up to 78% lower than those of the ZN treatment, and there was a significant difference between the two. The cumulative N2O emissions of the ZJ treatment were 64.52% lower than those of the ZN treatment, and there was a significant difference between the two ( Figure 6 and Figure 7 ).

[0066] 4. Effect of Pseudomonas sp.K33 on NO3 in farmland soil - -N content and the effect on maize plant height

[0067] NO3 - -N data collection: Soil at different depths (0-10 cm, 15-30 cm) was collected on the 8th day after fertilization in the embodiment and comparative example. 10 g of fresh soil was weighed into a centrifuge tube and 50 mL of 1 mol / L KCl solution was added. After shaking on a shaker for 1 hour, the filtrate was filtered with filter paper to obtain the filtrate. The NO3 in the filtrate was determined by ultraviolet spectrophotometer. - -N concentration.

[0068] Data collection of corn plant height and fresh weight: When the corn was 60 days old, the plant height and fresh weight of corn in ZJ and ZN treatments were measured. Ten corn plants were randomly selected from each treatment to measure the plant height and fresh weight of corn.

[0069] Data analysis: 8 days after fertilization, ZJ treatment 15-30 cm NO3 - -N content was 13% lower than that of the ZN treatment (P<0.05) (see Table 1 for details); the T-test test in R language was used to perform statistical analysis on the fresh weight index of corn plant height, and it was found that after 60 days of growth, the plant height of corn in the ZJ treatment was lower than that in the ZN treatment, and the fresh weight of corn in the ZJ treatment was higher than that in the ZN treatment (see Table 2 for details). This is because the nitrogen content in the soil treated with ZN in the comparative example is excessive, resulting in excess nutrition of the corn in the comparative example and thus leggy plant growth. However, in the soil treated with ZJ in the embodiment, the Pseudomonas sp.K33 strain converts excess nitrate in the soil into N2 for emission, thereby avoiding the absorption of excess nutrition by corn, and further making the fresh weight of the corn in the embodiment heavier, indicating that the consumption of excess nitrate will not only not affect the normal growth of crops, but will also promote plant growth to a certain extent.

[0070] Table 1

[0071]

[0072] Table 2

[0073]

[0074] In summary, Pseudomonas sp.K33, as an efficient N2O reduction strain, can significantly reduce the emission of N2O after the application of biogas slurry, and has a certain effect on reducing the accumulation of nitrate in farmland soil without affecting crop growth. The application of this strain can reduce greenhouse gas emissions from farmland and provide technical support for the development of environmentally friendly biogas fertilizers.

Claims

1. A strain for reducing N2O emissions from soils with large amounts of biogas slurry, characterized in that: The strain is Pseudomonassp.K33, which is deposited in the China Type Culture Collection, with the deposit address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the deposit number being CCTCC M 20242032, the deposit date being September 23, 2024, and the 16S rRNA gene sequence being shown in SEQ ID NO.

1.

2. The method for using the strain for reducing N2O emission in soil with large amounts of biogas slurry according to claim 1, characterized in that: The following steps are involved: The Pseudomonas sp.K33 strain is prepared into a Pseudomonas sp.K33 bacterial agent, and then the Pseudomonas sp.K33 bacterial agent is inoculated into biogas slurry, and then the biogas slurry is applied to farmland soil.

3. The method for using a strain to reduce N2O emissions from soils with large amounts of biogas slurry according to claim 2, characterized in that: The preparation method of the Pseudomonas sp.K33 bacterial agent specifically comprises the following steps: S1, taking the feces and digestate after anaerobic fermentation and placing it in a culture bottle A and sealing the culture bottle A, then replacing the air in the culture bottle A with helium, and then adding N2O to the culture bottle A for enrichment culture; S2. After the enrichment culture is completed, the enrichment culture sample and sterile water are added to a sterile centrifuge tube, and cultured at 26-30°C for 25-35 minutes. After standing, the supernatant is taken for gradient dilution and spread on the TSA medium solid respectively, and then the TSA medium solid coated with the supernatant is placed in an air bag filled with N2O concentration of 4-6% and sealed for purification to obtain the strain Pseudomonas sp.K33; S3, take another culture bottle B, add TSA medium liquid to culture bottle B, inoculate OD 600 The Pseudomonas sp.K33 strain was 0.4-0.6, the culture bottle B was sealed and cultured at 26-30℃. 600 When the pH value is 0.8-1, the culture is terminated to obtain the bacterial solution of Pseudomonas sp.K33; S4. Centrifuge the Pseudomonas sp. K33 bacterial solution in culture bottle B at 3800-4200 r / min, discard the supernatant, and then resuspend the bacterial cells in sterile 1× phosphate buffer to obtain a Pseudomonas sp. K33 bacterial agent.

4. The method for using a strain to reduce N2O emissions from soils with large amounts of biogas slurry according to claim 3, characterized in that: In the culture bottle A, 0.22-26 mL N2O is added for every gram of fecal digestate.

5. The method for using a strain to reduce N2O emissions from soils with large amounts of biogas slurry according to claim 3, characterized in that: In step S2, the solid content of the TSA culture medium per liter includes: 12-18 g of tryptone, 4-6 g of soy peptone, 4-6 g of sodium chloride, and 12-18 g of agar.

6. The method for using a strain to reduce N2O emissions from soils with large amounts of biogas slurry according to claim 3, characterized in that: In step S3, each liter of the TSA culture medium liquid includes: 12-18 g of tryptone, 4-6 g of soy peptone, and 4-6 g of sodium chloride.

7. The method for using a strain to reduce N2O emissions from soils with large amounts of biogas slurry according to claim 2, characterized in that: The mass ratio of the Pseudomonas sp.K33 bacterial agent to the biogas slurry is 1-2:1000, and then the biogas slurry is applied to the farmland soil at a dosage of 4-5 cubic meters per mu.

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