New application of bacillus subtilis WJL-2

Abstract

The invention discloses a novel application of bacillus subtilis WJL-2, and relates to the technical field of microorganisms. The invention discloses a novel application of bacillus subtilis WJL-2. The preservation number of the bacillus subtilis WJL-2 is GDMCC65111, and the preservation number of the bacillus subtilis WJL-2 is GDMCC65111. The new application specifically comprises the following steps: improving soil, regulating and controlling the content of total nitrogen and organic carbon in the soil, regulating and controlling the activity of amylase / urease / cellulase in the soil, regulating and controlling the content of ammonium nitrate nitrogen in the soil, regulating and controlling the mineralization rate of the soil, regulating and controlling the nitration rate of the soil and regulating and controlling the ammoniation rate of the soil. After the strain WJL-2 is inoculated into the soil, the total nitrogen and organic carbon content of the soil is reduced, and the C / N is reduced; the activity of soil amylase is reduced, and the activity of urease and cellulase is increased; the content of ammonium nitrogen in the soil is in a trend of firstly increasing and then decreasing, and the content of nitrate nitrogen is continuously increased; the mineralization rate of soil organic nitrogen is accelerated, the ammoniation rate is in a trend of increasing first and then decreasing, and the nitration rate is in a trend of decreasing first and then increasing.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and more specifically, relates to a new use of Bacillus subtilis WJL-2. Background Art

[0002] my country has broad research and application prospects in the treatment and utilization of organic nitrogen, ammonium nitrogen and nitrate nitrogen. As an important form of nitrogen in the soil, organic nitrogen plays an important role in agricultural production, while ammonium nitrogen and nitrate nitrogen are nitrogen forms that can be directly absorbed and utilized by plants. In terms of organic nitrogen mineralization technology, my country mainly converts organic nitrogen into ammonium nitrogen and nitrate nitrogen that can be absorbed by plants through the action of microorganisms. This process involves a variety of microorganisms, including Pseudomonas, Bacillus, Streptomyces, etc., which play a key role in the soil.

[0003] Although there are many technologies for the mineralization of organic nitrogen and the treatment of ammonium nitrogen and nitrate nitrogen, these technologies still face some challenges in practical application. For example, the low efficiency of organic nitrogen mineralization, unstable microbial activity, and slow degradation rate limit the widespread application of the technology. In the treatment of ammonium nitrogen and nitrate nitrogen, although the existing microbial electrochemical methods can recover ammonia, their efficiency and stability still need to be improved, and the operating conditions are relatively high. In addition, the strains also have problems such as low enzyme activity, poor stability, and slow mineralization and nitrification rates, which significantly affect the efficiency and application scope of organic nitrogen mineralization. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide an organic nitrogen mineralizing bacterium WJL-2. The technical problem to be solved by the present invention is to provide an application of an organic nitrogen mineralizing bacterium WJL-2 for soil improvement.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] Application of Bacillus subtilis WJL-2 in soil improvement, wherein the deposit number of the Bacillus subtilis WJL-2 is GDMCC65111.

[0007] The invention discloses an application of Bacillus subtilis WJL-2 in regulating the total nitrogen content in soil. The deposit number of the Bacillus subtilis WJL-2 is GDMCC65111.

[0008] Application of Bacillus subtilis WJL-2 in regulating the organic carbon content in soil, the Bacillus subtilis WJL-2 deposit number is: GDMCC65111

[0009] The invention discloses an application of Bacillus subtilis WJL-2 in regulating the activity of amylase in soil. The deposit number of the Bacillus subtilis WJL-2 is GDMCC65111.

[0010] Application of Bacillus subtilis WJL-2 in regulating urease activity in soil, wherein the Bacillus subtilis WJL-2 has a deposit number of GDMCC65111.

[0011] The invention discloses an application of Bacillus subtilis WJL-2 in regulating cellulase activity in soil. The deposit number of the Bacillus subtilis WJL-2 is GDMCC65111.

[0012] The invention discloses an application of Bacillus subtilis WJL-2 in regulating the content of ammonium nitrate nitrogen in soil. The deposit number of the Bacillus subtilis WJL-2 is GDMCC65111.

[0013] The application of Bacillus subtilis WJL-2 in regulating the mineralization rate of soil, the Bacillus subtilis WJL-2 has a deposit number of GDMCC65111.

[0014] The invention discloses an application of Bacillus subtilis WJL-2 in regulating the nitrification rate of soil. The deposit number of the Bacillus subtilis WJL-2 is GDMCC65111.

[0015] The invention discloses an application of Bacillus subtilis WJL-2 in regulating the ammonium rate of soil. The deposit number of the Bacillus subtilis WJL-2 is GDMCC65111.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1) After the strain WJL-2 was inoculated in the soil, the total nitrogen content of the soil decreased slightly, and decreased to 1.11 g / kg on the seventh day; the organic carbon content gradually decreased, and the C / N also decreased with time.

[0018] 2) After the strain WJL-2 was inoculated in the soil, the soil amylase activity first increased and then slightly decreased over time, reaching a peak of 13.65 U / g on the 5th day and a final value of 13.47 U / g.

[0019] 3) After the strain WJL-2 was inoculated in the soil, the soil urease activity increased slightly from 0 to 3 days, and began to decline continuously after 3 days, decreasing to 563.78 U / g on the 7th day.

[0020] 4) After the strain WJL-2 was inoculated in the soil, the soil cellulase activity reached the highest activity on the 3rd day, reaching 3.68 U / g; the control group reached the highest activity of 3.41 U / g on the 5th day.

[0021] 5) After the strain WJL-2 was inoculated in the soil, the ammonium nitrogen in the soil showed a trend of first rising and then falling, rising from 7.45 mg / L on the 0th day to 9.78 mg / L on the 3rd day, reaching a peak, and then falling in the next four days, falling to 9.23 mg / L on the 7th day. The nitrate nitrogen content continued to rise, from 1.75 mg / L on the 0th day to 3.54 mg / L on the 7th day.

[0022] 6) After the strain WJL-2 was inoculated in the soil, the mineralization rate of soil organic nitrogen accelerated; the ammonification rate showed a trend of first increasing and then decreasing; the nitrification rate gradually decreased from 0 to 3 days, gradually increased after 3 days, and finally reached 27.73 on the 7th day, while the nitrification rate of the blank group fluctuated between 26.18% and 30.59%. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the change diagram of total nitrogen in soil of the soil culture experiment of strain WJL-2;

[0024] Figure 2 This is the change diagram of soil organic carbon in the soil culture experiment of strain WJL-2;

[0025] Figure 3 This is the change diagram of soil carbon-nitrogen ratio in soil culture experiment of strain WJL-2;

[0026] Figure 4 Standard curve diagram for measuring amylase;

[0027] Figure 5 This is the change diagram of soil amylase activity in soil culture experiment of strain WJL-2;

[0028] Figure 6 The standard curve diagram for measuring urease;

[0029] Figure 7 This is the change diagram of soil urease activity in soil culture experiment of strain WJL-2;

[0030] Figure 8 The standard curve diagram for measuring cellulase;

[0031] Fig. 9 This is the change chart of soil cellulase activity in soil culture experiment of strain WJL-2;

[0032] Fig.10 This is the standard curve of soil ammonium nitrogen in the soil culture experiment of strain WJL-2;

[0033] Fig.11 This is the standard curve of soil nitrate nitrogen in the soil culture experiment of strain WJL-2;

[0034] Fig.12This is the change diagram of soil ammonium nitrogen in the soil culture experiment of strain WJL-2;

[0035] Fig.13 This is the change diagram of soil nitrate nitrogen in the soil culture experiment of strain WJL-2;

[0036] Fig.14 This is the change diagram of soil mineralization rate in soil culture experiment of strain WJL-2;

[0037] Fig.15 This is the change diagram of soil ammoniation rate in soil culture experiment of strain WJL-2;

[0038] Fig.16 This is a graph showing the changes in soil nitrification rate in the soil culture experiment of strain WJL-2. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0040] The Bacillus subtilis WJL-2 used in this application is the Bacillus subtilis WJL-2 disclosed in Chinese Patent 202411627715X, which is deposited in Guangdong Provincial Microbiological Culture Collection Center, with a deposit number of GDMCC65111, a deposit date of September 9, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0041] The test soil used in this application was collected from the tobacco planting base in Xinning County, Hunan Province.

[0042] Example 1

[0043] 1. Preparation of bacterial suspension

[0044] The obtained WJL-2 single colony was inoculated into a 250 mL conical flask containing 100 mL LB liquid medium, placed in a shaker at 28°C and 220 r / min for 48 h, and then centrifuged at 4000 r / min to obtain the bacterial cells. The bacterial solution OD was adjusted with physiological saline. 600 =1.8, and obtain the bacterial suspension for soil cultivation test.

[0045] 2. Inoculate soil

[0046] The collected surface soil (0-20cm) was passed through a 2mm sieve for later use. 200g of soil was filled into each of the four prepared small flower pots, one of which was used as the control group, and 2ml of the cultured Bacillus suspension was added to the other three pots as parallel repeated treatments. Adjust to 60% water holding capacity, seal with sealing film, and place it in an incubator at 25°C for culture. A random test of pre-culture elimination temperature was carried out for the first three days, and the formal culture stage began on the fourth day. During the formal culture stage, samples were taken on the 0th, 1st, 3rd, 5th, and 7th days of culture to determine the total nitrogen, organic carbon, ammonium nitrogen, nitrate nitrogen content of the soil, as well as urease, amylase, and cellulase, and the mineralization rate, ammoniation rate, and, where day 0 was the initial value at the end of pre-culture.

[0047] 3. Determination of soil organic carbon and total nitrogen

[0048] Soil organic carbon and total nitrogen were measured using a carbon-nitrogen analyzer on days 0, 1, 3, 5, and 7, respectively.

[0049] The results are as follows Figure 1-3 As shown, the total nitrogen and organic carbon contents of the control group did not change significantly over time. The total nitrogen content of the group treated with strain WJL-2 decreased slightly, and decreased to 1.11 g / kg ( Figure 1 ); the organic carbon content gradually decreases ( Figure 2 ), and C / N also decreases with time ( Figure 3 ).

[0050] 4. Determination of enzyme activity

[0051] 1) Amylase activity

[0052] Air-dry the fresh soil sample and pass it through a 30-50 mesh sieve. Preheat the microplate reader for 30 minutes and adjust the wavelength to 540 mm. Prepare the reagent 2 powder in the kit (Soil Amylase S-AMS Kit of Nanjing Mofan Biotechnology Co., Ltd.) into an application solution, and add 10 mg of the standard powder to 1.38 mL of water to prepare a 20 μmol / mL standard, which is then diluted with distilled water to 2, 1, 0.8, 0.6, and 0.4 μmol / mL standard solutions. Prepare test tubes and control tubes that match the number of samples. Add 0.05 g of soil sample, 10 μL of toluene, and 100 μL of reagent 1 to each test tube and control tube. Add 100 μL of reagent 2 application solution to the test tube and 100 μL of distilled water to the control tube. Mix well, incubate at 37° for 24 hours, mix well, centrifuge at 10,000 rpm for 10 minutes at room temperature, and take the supernatant for later use. Take 60mL of supernatant from the test tube and control tube, add 140μL of reagent 3 and set aside. Prepare blank tubes and standard tubes, add 60μL of the above standard solutions of each concentration into the standard tube, add 60μL of distilled water into the blank tube, and add 140μL of reagent 3 into both the standard tube and the blank tube.

[0053] After the test tube, control tube, blank tube and standard tube are fully mixed, boil in water for 10 min, cool on ice, and measure the absorbance at 540 nm in a 1 mL glass cuvette, record as A 测定管 , A 对照管 , A 空白管 , A 标准管 , calculate ΔA 测定 =A 测定管 -A 对照管 , ΔA 标准 =A 标准管 -A 空白管 .

[0054] According to the concentration (x, μmol / L) and absorbance ΔA of the standard tube 标准 (y, ΔA 标准 ), and a standard curve was established. The standard curve of amylase activity was y = 1.3429x-0.2797, and the correlation coefficient was R 2 =0.9914, which proves that there is a good linear relationship between amylase activity and absorbance value. The amylase activity of the sample can be calculated by the absorbance value of the sample through the equation ( Figure 4 ). According to the standard curve, ΔA 测定 (y, ΔA 测定 ) into the formula to calculate the sample concentration (x, μmol / L)

[0055] The production of 1 μmol of reducing sugar per gram of soil sample per day was defined as one soil amylase activity.

[0056] S-AMS(U / g)=x×V 反总 ÷W÷T=0.42×x÷W.

[0057] V 反总 : total volume of enzymatic reaction, 0.42 mL; W: soil sample mass, g; T: reaction time, 1 d.

[0058] The results are as follows Figure 5 As shown, the amylase activity of the control group did not change significantly, fluctuating in the range of 10.56U / g-10.96U / g. The amylase activity of the group treated with strain WJL-2 first increased and then slightly decreased over time, reaching a peak of 13.65U / g on the 5th day, and the final value was 13.47U / g.

[0059] 2) Urease activity

[0060] Air-dry the fresh soil sample naturally or in a 37°C oven, and pass it through a 30-50 mesh sieve. Preheat the spectrophotometer for more than 30 minutes, adjust the wavelength to 630nm, and set it to zero with distilled water. Take an appropriate amount of air-dried soil sample and mix it with toluene. After adding reagents 1 and 2, let it stand at room temperature for 15 minutes, then add reagent 3, mix well and place it in a 37°C water bath or constant temperature incubator for 24 hours. After the incubation, dilute the supernatant and perform a color reaction. After adding reagents 4 and 5, let it stand at room temperature for 20 minutes, dilute it with distilled water and perform a spectrophotometer at a wavelength of 630nm to calculate the absorbance value. Finally, the soil urease activity, that is, the amount of NH3-N produced per gram of air-dried soil per day, is calculated based on the standard curve.

[0061] Prepare the standard curve by diluting 1 mg / mL nitrogen standard solution with distilled water to 0, 1, 2, 4, 6, 8, 10 μg / mL nitrogen standard solution. Mix well, set the wavelength to 630 nm, 1 cm optical path, adjust to zero with distilled water, and measure the absorbance value of each tube. The standard curve of urease activity is y=16.317x-0.4446, and the correlation coefficient is R 2 =0.9989, which proves that there is a good linear relationship between urease activity and absorbance value. The urease activity of the sample can be calculated by the equation using the absorbance value of the sample ( Figure 6 ).

[0062] One unit of enzyme activity is 1ug NH3-N produced per gram of air-dried soil per day.

[0063] S-UE(U / g soil sample)=(Y 测定管 -Y 对照管 -Y 基质管 )×V 反总 ÷W÷T.

[0064] T: reaction time, 1d; V 反总 : total volume of enzymatic reaction, 1.5 mL; W: sample mass, 0.1 g.

[0065] The results are as follows Figure 7 As shown in the figure, the urease activity of the soil with strain WJL-2 increased slightly from 0 to 3 days, and then began to decline continuously after 3 days, decreasing to 563.78 U / g on the 7th day. The urease activity of the control group did not change significantly.

[0066] 3) Cellulase activity

[0067] Air dry the fresh soil sample naturally or in a 37°C oven, and pass through a 30-50 mesh sieve. Prepare a corresponding number of test tubes, control tubes, and a soilless matrix tube, add 0.05g of soil sample to the test tube and control tube respectively, then add 25μL of toluene to the three tubes, shake and mix, make all the soil samples wet, and let stand at room temperature for 15min. Then add 500μL to the test tube and soilless matrix tube, and 500μL distilled water to the control tube, mix, incubate at 37° for 24h, mix, centrifuge at 10000 rpm for 10min at room temperature, and take the supernatant for use. Take 50μL of the diluted supernatant from the control tube, test tube, and soilless matrix tube, then add 150μL of reagent 2 to each, mix in a boiling water bath for 5min, and cool with running water. Then add 500μL of distilled water respectively. After mixing, draw 200μL into the ELISA plate, adjust the wavelength to 540nm, and measure the absorbance value of each tube.

[0068] Dilute the 10 mg / mL standard with distilled water to 0, 0.2, 0.4, 0.6, 0.8, 1 mg / mL standard to prepare the standard curve. Add 50 μL of different concentrations of standard and reagent 2 to each standard tube, mix well in a boiling water bath for 5 minutes, cool with running water, and add 500 μL of distilled water for measurement. According to the standard curve, substitute the absorbance value into the standard curve to calculate the concentration Y (mg / mL) in the supernatant. The standard curve of cellulase activity is y=0.5697x+0.0048, and the correlation coefficient R 2 =0.9969, which proves that there is a good linear relationship between cellulose activity and absorbance value. The absorbance value of the sample can be used to calculate the cellulase activity of the sample through the equation ( Figure 8 ).

[0069] One unit of enzyme activity is 1 mg of reducing sugar produced per gram of air-dried soil per day.

[0070] Determination tube content (U / g) = (Y 测定管 -Y 无土基质管 )×N×V 反总 ÷W÷T.

[0071] Control tube content (U / g) = Y 对照管 ×N×V 反总 ÷W÷T.

[0072] S-CL (U / g soil sample) = content in test tube - content in control tube; N: dilution multiple of supernatant; T: reaction time, 1d; V 反总 : total volume of reaction system, 0.5 mL; W: sample mass, 0.05 g.

[0073] The results are as follows Fig. 9As shown, the cellulase activity of the treatment group with strain WJL-2 was the highest on the 3rd day, reaching 3.68 U / g; the cellulase activity of the control group reached the highest on the 5th day, reaching 3.41 U / g.

[0074] 5. Determination of ammonium nitrate nitrogen content

[0075] Weigh 10.0g of the test soil into a 500mL polyethylene bottle with a screw cap, add 100mL of potassium chloride extract, shake at 200r / min at room temperature for 1h, and filter and collect the filtrate. Use AA3 continuous flow analyzer to determine the concentrations of ammonium nitrogen and nitrate nitrogen in the extract of the experimental group. Determine the ammonium nitrogen content with an enzyme marker: take 100μL of the test solution, add 50μL of buffer and 50μL of phenol solution, and after 1h of color reaction in the dark, measure the absorbance at a wavelength of 625nm. Determine the nitrate nitrogen content with an enzyme marker: take 40μL of the test solution, add 40μL of sodium hydroxide solution and 40μL of hydrazine sulfate solution, seal the enzyme labeling plate with a sealing film, react for 20min, add 80μL of buffer and 40μL of color developer, and measure the absorbance at a wavelength of 500nm.

[0076] Ammonium nitrogen standard curve: Take 0, 0.1, 0.2, 0.3, 0.4, 0.5 mL of ammonium chloride stock solution respectively, and dilute to 50.0 mL with water, i.e., the concentration of ammonium nitrogen standard solution is 0, 0.2, 0.4, 0.6, 0.8, 1.0 mg / L. The ammonium nitrogen standard curve is y=0.3519x+0.0079, R 2 = 0.9965, and the absorbance can be entered to obtain the nitrate nitrogen and ammonium nitrogen content of each sample ( Fig.10 ).

[0077] Nitrate nitrogen standard curve: Take 0, 1, 2, 3, 4, 5 mL of potassium nitrate stock solution respectively, and dilute to 50.0 mL with water, i.e., nitrate nitrogen standard solution with concentrations of 0, 2, 4, 6, 8, 10 mg / L. The nitrate nitrogen standard curve is y=0.0791x-0.0171, R 2 = 0.9984, and the absorbance can be entered to obtain the nitrate nitrogen and ammonium nitrogen content of each sample ( Fig.11 ).

[0078] The results are as follows Fig.12 and Fig.13 As shown in the figure, there was no significant change in the nitrate nitrogen and ammonium nitrogen in the control group. The ammonium nitrogen in the treatment group with strain WJL-2 showed a trend of first rising and then falling, rising from 7.45 mg / L on the 0th day to 9.78 mg / L on the 3rd day, and then falling in the next four days, falling to 9.23 mg / L on the 7th day ( Fig.12 ). The nitrate nitrogen content continued to rise, from 1.75 mg / L on the 0th day to 3.54 mg / L on the 7th day ( Fig.13).

[0079] 6. Mineralization rate, ammoniation rate and nitrification rate

[0080] Inorganic nitrogen content ≈ nitrate nitrogen + ammonium nitrogen content.

[0081] Mineralization rate = (soil inorganic nitrogen content on day 7 - soil inorganic nitrogen content on day 0) / time.

[0082] Ammoniation rate = (ammonium nitrogen content / inorganic nitrogen content) × 100%.

[0083] Nitrification rate = (nitrate nitrogen content / inorganic nitrogen content) × 100%.

[0084] The results are as follows Fig.14 As shown in Figure 2, after seven days of soil culture experiments, the mineralization rate of the blank group soil was calculated to be 0.06 mg·L -1 / d, while the treatment group with Bacillus strain WJL-2 was 0.51 mg·L -1 / d. The results showed that strain WJL-2 promoted the mineralization rate of soil organic nitrogen.

[0085] The results are as follows Fig.15 As shown, the ammoniation rate of the treated group showed a trend of first increasing and then decreasing, reaching a peak of 82.04% on the 3rd day, while there was no obvious trend in the blank group.

[0086] The results are as follows Fig.16 As shown, the nitrification rate of the treatment group gradually decreased from 0 to 3 days, gradually increased after the 3rd day, and finally reached 27.73 on the 7th day. The nitrification rate of the blank group fluctuated between 26.18% and 30.59%.

[0087] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. Application of Bacillus subtilis WJL-2 in soil improvement, the deposit number of the Bacillus subtilis WJL-2 is: GDMCC65111.

2. Application of Bacillus subtilis WJL-2 in regulating the total nitrogen content in soil. The deposit number of the Bacillus subtilis WJL-2 is: GDMCC65111.

3. Application of Bacillus subtilis WJL-2 in regulating the organic carbon content in soil, the Bacillus subtilis WJL-2 has a deposit number of GDMCC65111.

4. Application of Bacillus subtilis WJL-2 in regulating the activity of amylase in soil. The deposit number of the Bacillus subtilis WJL-2 is GDMCC65111.

5. Application of Bacillus subtilis WJL-2 in regulating urease activity in soil, the Bacillus subtilis WJL-2 has a deposit number of GDMCC65111.

6. Application of Bacillus subtilis WJL-2 in regulating cellulase activity in soil, the deposit number of the Bacillus subtilis WJL-2 is: GDMCC65111.

7. Application of Bacillus subtilis WJL-2 in regulating the content of ammonium nitrate nitrogen in soil. The deposit number of the Bacillus subtilis WJL-2 is GDMCC65111.

8. Application of Bacillus subtilis WJL-2 in regulating the mineralization rate of soil, the Bacillus subtilis WJL-2 has a deposit number of GDMCC65111.

9. Application of Bacillus subtilis WJL-2 in regulating soil nitrification rate, the Bacillus subtilis WJL-2 has a deposit number of GDMCC65111.

10. Application of Bacillus subtilis WJL-2 in regulating the ammonium rate of soil, wherein the deposit number of the Bacillus subtilis WJL-2 is GDMCC65111.

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