A urease inhibitor-producing microbial strain and its field application

Abstract

The application discloses a urease inhibitor-producing microbial strain and field application thereof, and belongs to the technical field of microorganisms.The microbial strain of the application is Priestia endophytica GW16 with the preservation number of CCTCC NO: M 2022440, the strain has high activity in producing urease inhibitors, and when the strain is combined with nitrogen fertilizer and applied in the field, the soil nitrogen fertilizer utilization rate can be improved, and the emission of greenhouse gases N2O and CH4 in the soil can be reduced.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a microbial strain that produces urease inhibitors and its application in the field. Background Technology

[0002] In my country's agricultural chemical nitrogen application, ammonium bicarbonate and urea are the main types of nitrogen fertilizers. Nationwide, corn has the highest demand for urea, followed by rice and wheat. Urea is a physiologically neutral fertilizer with many advantages: high nitrogen content (45%-46%); low cost; easy solubility in water and easy disposal (not likely to cause fire or explosion). It is also suitable for producing compound fertilizers; it can be applied to the soil in solid or liquid form and can be used as a foliar fertilizer for some crops. However, there are also several problems with the application of urea fertilizer. The main issue is that it rapidly hydrolyzes into ammonium carbonate under the action of soil urease, leading to an increase in soil pH and the accumulation of ammonia. This can damage germinating seedlings and young plants, producing toxic nitrites and ammonia; and causing urea nitrogen to be lost in the form of ammonia gas. Furthermore, the rapid hydrolysis of urea caused by soil urease increases the concentration of ammonia and pH in the soil, slowing down the process of microbial oxidation of nitrite to nitrate, leading to the accumulation of nitrous oxide (N2O) which is harmful to crops. N2O is a significant greenhouse gas, with agricultural activities accounting for more than half of the total N2O emissions from human activities. Fertilization is a major source of N2O emissions in agricultural systems, and urea is a widely used fertilizer in agricultural production, accounting for approximately 66% of global chemical nitrogen fertilizer emissions. Therefore, improving the utilization efficiency of urea nitrogen fertilizer is crucial for N2O emission reduction.

[0003] Urease inhibitors can suppress urease activity, thereby slowing down the hydrolysis rate of urea and the release rate of ammonia, thus reducing nitrogen loss caused by ammonia volatilization and nitrification. The effectiveness of urease inhibitors is greatly influenced by environmental conditions, such as soil pH, moisture, soil temperature, soil aeration, soil fertility, and soil type. Different types of urease inhibitors are suitable for different environmental conditions. NBPT (butyl thiophosphate triamine) is least affected by soil pH and is suitable for use in both acidic and alkaline soils. PPD (phenylphosphamide diamine), while a relatively effective urease inhibitor, is prone to decomposition into phenol, which is a less effective urease inhibitor, significantly reducing its inhibition efficiency. Soil urease activity decreases with increasing hydroquinone (HQ) dosage. Hydroquinone is less affected by soil environmental factors such as pH and aeration, but HQ is less effective than NBPT and PPD under well-aerated conditions, and less effective than PPD under anaerobic conditions. Plant-derived urease inhibitors are relatively safe, but plants have long growth cycles, raw material sources are unstable, extraction processes are complex, large-scale production is costly and inefficient, and their inhibitory effect on urease is relatively low. Different urease inhibitors are affected by different environmental factors, limiting their inhibitory effects. Therefore, it is necessary to screen for the most suitable urease inhibitors based on different soil conditions. Summary of the Invention

[0004] The purpose of this invention is to provide a microbial strain that can produce urease inhibitors and its application in the field to reduce the emission of greenhouse gases such as N2O and CH4.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention isolates and identifies a microbial strain that can produce urease inhibitors from soil. This strain is deposited at the China Center for Type Culture Collection on April 21, 2022, and is classified as Priestiaendophytica GW16 with accession number CCTCC NO: M 2022440.

[0007] The strain Priestia endophytica GW16 provided by this invention was isolated and screened from soil and has the activity of producing urease inhibitors. Its 16S rDNA sequence is shown in SEQ ID NO.1.

[0008] The screening method for this strain specifically includes the following steps:

[0009] 1. Take 1g of soil from the vegetable growing area and place it in 100mL of sterile physiological saline to make a bacterial suspension. After serial dilution, spread it on a plate primary screening medium and incubate at 37℃ for 24h. The plate primary screening medium consists of: 20g urea, 10g sucrose, 10g peptone, 3g beef extract, 0.2g phenol red, 5g sodium chloride and 20g agar, 1000mL deionized water, pH 6.2.

[0010] 2. Pick undiscolored colonies from the colonies obtained in step 1 and transfer them to beef extract peptone agar medium for streaking incubation for 24 hours. The composition of beef extract peptone agar medium is: 3g beef extract, 10g peptone, 5g NaCl, 20g agar, 1000mL deionized water, pH 7.4-7.6.

[0011] 3. Pick colonies with pinkish tinges from the colonies obtained in step 1 and transfer them to beef extract peptone agar medium for streaking incubation for 24 hours. The composition of beef extract peptone agar medium is: 3g beef extract, 10g peptone, 5g NaCl, 20g agar, 1000mL deionized water, pH 7.4-7.6.

[0012] 4. From the colonies cultured in step 2, pick a single colony and inoculate it into beef extract peptone liquid medium (the composition of beef extract peptone liquid medium is: 3g beef extract, 10g peptone, 5g NaCl, 1000mL deionized water, pH 7.4-7.6), and incubate at 37℃ and 200rpm for 24h to obtain the fermentation broth of each colony.

[0013] 5. From the colonies cultured in step 3, pick a single colony and inoculate it into beef extract peptone liquid medium (the composition of beef extract peptone liquid medium is: 3g beef extract, 10g peptone, 5g NaCl, 1000mL deionized water, pH 7.4-7.6), and culture at 37℃ and 200rpm for 24h to obtain the fermentation broth of the urease strain.

[0014] 6. Take 1 mL of each of the fermentation broths obtained in step 4 and place them in a 1.5 mL Eppendorf tube. Centrifuge at 12000 rpm for 3 min and take the supernatant to prepare the sample solution to be selected.

[0015] 7. Take 1 mL of the fermentation broth obtained in step 5 and place it in a 1.5 mL Eppendorf tube. Centrifuge at 12000 rpm for 3 min and take the supernatant to prepare crude urease extract.

[0016] 8. Mix the candidate sample solution obtained in step 6 with the crude urease extract obtained in step 7, detect urease activity, and screen out the strain Priestia endophytica GW16 that produces urease inhibitors.

[0017] The strain Priestia endophytica GW16 provided by this invention has the activity of producing urease inhibitors, which can be used to inhibit soil urease activity.

[0018] The strain *Priestia endophytica* GW16 provided by this invention, when formulated into a microbial agent and applied to soil, can significantly reduce N2O and CH4 emissions. The microbial agent includes fermentation broth, powder, and granules. The method for preparing the microbial powder from strain *Priestia endophytica* GW16 is as follows: first, liquid deep fermentation is performed; then, the resulting fermentation broth is continuously centrifuged; finally, the precipitate obtained by centrifugation is spray-dried. Using a process combining liquid deep fermentation, continuous centrifugation, and spray drying, the viable cell content of the resulting microbial powder from strain *Priestia endophytica* GW16 can reach 6.0 × 10⁻⁶. 10 CFU / g.

[0019] The strain Priestia endophytica GW16 provided by this invention can be applied to the soil in conjunction with nitrogen fertilizer to alleviate the adverse effects of urease in the soil on nitrogen fertilizer and improve nitrogen fertilizer utilization.

[0020] Advantages and beneficial effects of the present invention: When the fermentation broth of the urease inhibitor strain Priestia endophytica GW16 of the present invention is sprayed on fields where urea is applied, it can effectively reduce the emission of N2O and CH4, reduce nitrogen loss, improve nitrogen fertilizer utilization, reduce greenhouse gas emissions, and be more environmentally friendly.

[0021] Strain Preservation Information

[0022] Depository: China Center for Type Culture Collection; Address: Wuhan University, Wuhan, China; Date of deposit: April 21, 2022; Classification: Priestia endophytica GW16; Accession number: CCTCC NO: M2022440. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention.

[0024] Example 1

[0025] This embodiment provides a strain of Priestia endophytica GW16 that produces a urease inhibitor. This strain was obtained through the following screening steps:

[0026] 1. Screening of non-urease-producing strains

[0027] 1.1 Initial screening of non-urease-producing strains

[0028] Weigh 1g of soil from a vegetable growing area in Shouguang City, Shandong Province, and add it to a 250mL Erlenmeyer flask containing 100mL of sterile physiological saline and 15 glass beads. Shake at 100rpm for 5 minutes to prepare a bacterial suspension. Then, perform serial dilutions of this bacterial suspension using sterile physiological saline, with each dilution factor being 10. -1 10 -2 10 -3 10 -4 10 -5 10 -6 The concentration gradient is set at 10. -4 10 -5 10 -6 50 μL of bacterial suspension was spread onto five separate primary screening agar plates and incubated at 37°C for 24 h. The primary screening agar plates were prepared as follows: 20 g urea, 10 g sucrose, 10 g peptone, 3 g beef extract, 0.2 g phenol red, 5 g sodium chloride, and 20 g agar were added to 1000 mL of deionized water, mixed well, and the pH was adjusted to 6.2. The plates were then autoclaved at 121°C for 20 min.

[0029] 1.2 Culture of non-urease-producing strains

[0030] From the colonies obtained in step 1.1, select undiscolored colonies around the periphery and transfer them to beef extract peptone agar medium. Streak culture is performed, and the culture is incubated upside down at 37°C for 24 hours. Then, it is stored at 4°C for later use. The composition of beef extract peptone agar medium is: 3g beef extract, 10g peptone, 5g NaCl, 20g agar, 1000mL deionized water, pH 7.4-7.6.

[0031] 1.3 From the colonies cultured in step 1.2, pick a single colony and inoculate it into beef extract peptone liquid medium (the composition of beef extract peptone liquid medium is: 3g beef extract, 10g peptone, 5g NaCl, 1000mL deionized water, pH 7.4-7.6), and culture at 37℃ and 200rpm for 24h to obtain fermentation broth.

[0032] 1.4 Take 1 mL of each of the fermentation broths obtained in step 1.3 and place them in a 1.5 mL Eppendorf tube. Centrifuge at 12000 rpm for 3 min and take the supernatant to prepare the sample to be tested.

[0033] 2. Screening of urease-producing strains

[0034] 2.1 Initial screening of urease-producing strains

[0035] Pick colonies that have turned pink from the periphery of the colonies obtained in step 1.1 and transfer them to beef extract peptone agar medium for streak culture. The composition of beef extract peptone agar medium is: 3g beef extract, 10g peptone, 5g NaCl, 20g agar, 1000mL deionized water, pH 7.4-7.6.

[0036] 2.2 Culture of urease-producing strains

[0037] From the colonies cultured in step 2.1, pick a single colony and inoculate it into beef extract peptone liquid medium (the composition of beef extract peptone liquid medium is: 3g beef extract, 10g peptone, 5g NaCl, 1000mL deionized water, pH 7.4-7.6), and culture at 37℃ and 200rpm for 24h to obtain the fermentation broth of each colony.

[0038] 2.3 Secondary screening of urease-producing strains

[0039] Take 1 mL of each of the fermentation broths obtained in step 2.2 and place them in 1.5 mL Eppendorf tubes. Centrifuge at 12000 rpm for 3 min, and collect the supernatant to prepare a crude urease extract. The urease activity of the crude urease extract was detected using a Solarbio BC4110 kit. The activity of this crude urease extract was found to be 0.34 U / g.

[0040] 3. Screening of strains that produce urease inhibitors

[0041] Add 0.5 mL of urease-producing strain fermentation broth to a 1.5 mL Eppendorf tube, then add 0.5 mL of the test sample from the non-urease-producing strain obtained in step 1.4 above, mix well, and use this as the experimental group. Detect the urease activity of the experimental group using the Solarbio BC4110 kit. Use the test solution without the addition of the non-urease-producing strain as a control, measure its urease activity, and calculate the reduction value of urease activity to screen out the strain *Priestia endophytica* GW16, which possesses a urease inhibitor.

[0042] The 16S rDNA sequence of the urease inhibitor-producing strain *Priestia endophytica* GW16 is shown in SEQ ID NO. 1. This strain is deposited at the China Center for Type Culture Collection (CCCCC), Wuhan University, Wuhan, China, on April 21, 2022, and is classified as *Priestia endophytica* GW16 with accession number CCTCC NO: M2022440.

[0043] Example 2

[0044] This embodiment provides the inhibitory effect of Priestia endophytica GW16 fermentation broth on urease activity at 37°C.

[0045] 1. Fermentation of microbial urease inhibitors

[0046] A single colony of Priestia endophytica GW16 was picked and inoculated into beef extract peptone liquid medium (the composition of beef extract peptone liquid medium is: 3g beef extract, 10g peptone, 5g NaCl, 1000mL deionized water, pH 7.4-7.6), and cultured at 37℃ and 200rpm for 24h to obtain the fermentation broth.

[0047] 2. The urease used in this experiment was the crude urease solution obtained in step 2 of Example 1, with 1% NBPT (n-butyl thiophosphate) as a control. The experiment lasted for 7 days, with daily sampling to measure urease activity and calculate the percentage decrease in urease activity. The results are shown in Table 1. The results showed that the fermentation broth of GW16 inhibited urease activity by 15.34%. After 24 hours, the inhibition rate of urease by GW16 gradually decreased over time.

[0048] Table 1. Percentage of urease inhibition

[0049]

[0050] Example 3

[0051] This example demonstrates the application of Priestia endophytica GW16 as a microbial agent in the field to reduce N2O and CH4 emissions.

[0052] Priestia endophytica GW16 was inoculated into beef extract peptone liquid medium and cultured in a shaker at 200 rpm and 37°C for 24 h to obtain the seed culture.

[0053] The seed culture was inoculated into a 5L fermenter containing 3L of fermentation medium at a volume concentration of 1%-10%. The fermentation temperature was 37℃, the agitator speed was 100-200 rpm, the aeration rate was 0.5-1.0 vvm, and the incubation time was 24-48 hours. The fermentation medium composition (g / L) was: urea 20, sucrose 10, potassium chloride 5, magnesium sulfate 0.6, calcium chloride 0.6, disodium hydrogen phosphate 1, sodium dihydrogen phosphate 1, pH 6-8.

[0054] The obtained fermentation broth was evenly sprayed onto the fields using a small sprayer. The urea application rate was 14 kg / mu. Fields without microbial inoculant application served as negative controls, and fields sprayed with 1% NBPT served as positive controls. Each treatment was repeated three times. Gas samples were collected daily for five days after urea application, and then weekly thereafter to monitor the release of N2O and CH4 in the test fields. The results are shown in Tables 2 and 3. The results showed that N2O release peaked on the second day after urea application. The GW16 fermentation broth effectively inhibited N2O release, with an inhibition rate of 33.20%. CH4 release peaked on the 19th day after fermentation broth application. The fermentation broth effectively inhibited CH4 release, with an inhibition rate of 25.38% on the 19th day.

[0055] Table 2. N2O release rates for each treatment

[0056]

[0057] Table 3 CH4 release amounts for each treatment

[0058]

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A microbial strain that produces a urease inhibitor, characterized in that: The microbial strain mentioned is the one with the preservation number CCTCC NO: M 2022440. Priestia endophytica GW16.

2. The application of the microbial strain described in claim 1 in inhibiting soil urease activity.

3. The application of the microbial strain described in claim 1 in improving soil nitrogen fertilizer utilization.

4. The application of the microbial strain of claim 1 in reducing the emission of N2O and / or CH4 in soil.

5. The application according to any one of claims 2-4, characterized in that: The aforementioned microbial strains are formulated into inoculants and applied to the soil.

6. The application according to claim 5, characterized in that: The microbial agent is a fermentation broth, powder, or granules.

7. The application according to any one of claims 2-4, characterized in that: The aforementioned microbial strains are applied to the soil in conjunction with nitrogen fertilizer.

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