Method for measuring the contribution of ammonia-oxidizing microorganisms to soil nitrification

Abstract

The present invention discloses a method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification, comprising the following steps: Step 1, setting a determination method for nitrification potential; Step 2, distinguishing three microorganisms: in order to distinguish the contributions of three nitrifying functional microorganisms, ammonia oxidizing archaea (AOA), ammonia oxidizing bacteria (AOB) and complete nitrifying bacteria (comammox), the multi-inhibitor method is improved to distinguish them; thereby measuring NO2 at different soil suspension sampling time points. ‑ Content / NO3 ‑ The method of the present invention is used to accurately measure the contribution of ammonia-oxidizing archaea, ammonia-oxidizing bacteria, and complete nitrifying bacteria to soil nitrification.

Description

Technical Field

[0001] The present invention relates to a method for measuring the contribution of ammonia oxidizing archaea, ammonia oxidizing bacteria and complete nitrifying bacteria to soil nitrification, and the niche relationship among ammonia oxidizing archaea, ammonia oxidizing bacteria and complete nitrifying bacteria can be obtained by comparing the results. Background Art

[0002] Nitrification in soil usually refers to the process of converting ammonium nitrogen (NH4 + -N) is oxidized to nitrate nitrogen (NO3 - The process of nitrification (NH3-N) is catalyzed by microorganisms, with three key microorganisms being AOA (ammonia-oxidizing archaea), AOB (ammonia-oxidizing bacteria), and comammox (complete nitrifiers). Researchers have extensively studied the behavior of these three microorganisms in various ecological environments, but a definitive and accurate method for measuring their contribution to nitrification remains elusive. Currently, some common methods for measuring the contribution of these three microorganisms involve first measuring the total nitrification capacity of the soil, then inhibiting a specific microorganism with a single inhibitor and measuring the nitrification capacity of the remaining two microorganisms. This is then subtracted from the nitrification capacity of the corresponding microorganism without the inhibitor. However, this method often yields negative results in practice because it completely ignores intermicrobial interactions. When one microorganism is inhibited, the behavior of the other is unknown. In a mutually beneficial relationship, inhibition of one microorganism also inhibits the other, leading to skewed results. When two microorganisms are in a competitive relationship, inhibition of one can activate the other, resulting in skewed or even negative results. Therefore, the present invention is to improve the method for determining the contribution of AOA, AOB and comammox to soil nitrification, by incorporating the influence of interactions between microorganisms, so that the results are more accurate and the measurement method is more universal. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification.

[0004] In order to solve the above technical problems, the present invention provides a method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification, comprising the following steps:

[0005] Step 1: Set the nitrification potential measurement method (nitrification potential is used to represent soil nitrification capacity):

[0006] The soil to be tested was prepared into a soil suspension and cultured on a shaker (180 ± 20 rpm) for 24 h;

[0007] Set six sampling time points;

[0008] At each sampling time, the following operations were performed: 5 ml of the incubated soil suspension was taken and mixed with 5 ml of KCl solution (2 mol L -1 ) After mixing, shake and then centrifuge, the supernatant is filtered through 0.25mm filter membrane and then the NO2 is measured - and NO3 - content;

[0009] Description: It is necessary to determine the volumetric water content of the soil to be tested;

[0010] Step 2: Differentiation of three types of microorganisms:

[0011] In order to distinguish the contributions of three types of nitrifying microorganisms, ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB) and complete nitrifying bacteria (comammox), the multi-inhibitor method was improved to distinguish them.

[0012] The details are as follows:

[0013] 1) Add 100 ml of phosphate buffer to 10 g of the soil to be tested and then add NaClO3 to a final concentration of 10 mM; the soil suspension with NaClO3 added is named soil suspension I;

[0014] The soil suspension I was measured according to the method of step 1 at six sampling time points. - Content, obtain NO2 - The linear regression equation corresponding to the concentration and sampling time is used to obtain the slope R AOA+AOB ;

[0015] The soil suspension I was measured according to the method of step 1 at six sampling time points. - Content, obtain NO3 - The linear regression equation corresponding to the concentration and sampling time is used to obtain the slope R comammoxⅠ ;

[0016] 2) It includes the following two steps:

[0017] 2.1) Simvastatin was added to soil suspension I at a ratio of 12.5 mg of simvastatin per g of soil, and the resulting suspension was named simvastatin soil suspension;

[0018] The simvastatin soil suspension was measured according to the method of step 1 to determine the NO2 - Content, obtain NO2 - The linear regression equation corresponding to the concentration and sampling time is used to obtain the slope R AOB ;

[0019] 2.2) Add DMPP to the soil suspension I until the concentration of DMPP is 0.075 mM; the resulting suspension is named DMPP soil suspension;

[0020] The DMPP soil suspension was measured according to the method in step 1 to determine the NO2 at six sampling time points. - Content, obtain NO2 - The linear regression equation corresponding to the concentration and sampling time is used to obtain the slope R AOA ;

[0021] 3) Simvastatin and DMPP were added to soil suspension I simultaneously until the DMPP concentration reached 0.075 mM, with 12.5 mg of simvastatin added per g of soil. The resulting suspension was designated as simvastatin + DMPP soil suspension.

[0022] Simvastatin + DMPP soil suspension was measured according to the method of step 1 at six sampling time points. - Content, get NO3 - The linear regression equation corresponding to the concentration and sampling time is used to obtain the slope R comammoxⅡ ;

[0023] Step 3: Calculate the nitrification potential of the soil.

[0024] As an improvement to the method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification of the present invention:

[0025] The six sampling time points set in step 1 correspond to the 2nd, 4th, 8th, 20th, 22nd, and 24th hours of culture, respectively.

[0026] As a further improvement to the method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification of the present invention:

[0027] Np=R×24×(0.1+V) / m;

[0028] Np is the soil nitrification potential (mg kg -1 d -1 ), R is the growth rate of NO2-N or NO3-N content (mg L -1 h -1 ), 0.1 is the volume of phosphate buffer (L), V is the volume of water in the soil sample (L), and m is the mass of oven-dried soil (kg).

[0029] As a further improvement to the method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification of the present invention:

[0030] In phosphate buffer, NH4 + The concentration is 1.5mM, PO4 3- The concentration is 1 mM.

[0031] As a further improvement to the method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification of the present invention:

[0032] The KCl concentration in KCl solution is 2 mol L -1 .

[0033] As a further improvement to the method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification of the present invention:

[0034] When m = 10 g, V = 0.0007 L, Np = R × 241.68;

[0035] Np AOA+AOB =R AOA+AOB ×241.68;

[0036] Np comammoxⅠ =R comammoxⅠ ×241.68;

[0037] Np AOA =R AOA ×241.68;

[0038] Np AOB =R AOB ×241.68;

[0039] Np comammoxⅡ =R comammoxⅡ ×241.68.

[0040] illustrate:

[0041] Np AOA+AOB It represents the nitrification potential of AOA and AOB when nitrification occurs together, that is, it represents the nitrification potential of AOA and AOB when no inhibition is applied to ammonia oxidizing microorganisms;

[0042] Np AOA represents the nitrification potential of AOA when nitrification is carried out alone, that is, represents the nitrification potential of AOA when both AOB and comammox are inhibited;

[0043] Np AOB represents the nitrification potential of AOB when nitrification is carried out alone, that is, represents the nitrification potential of AOB when both AOA and comammox are inhibited;

[0044] comammox I represents the nitrification potential of comammox when ammonia-oxidizing microorganisms are not inhibited;

[0045] Comammox II represents the nitrification potential of comammox when both AOA and AOB are inhibited.

[0046] As a further improvement to the method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification of the present invention, in step 1: shaking at 220 rpm for 1 hour, and centrifuging at 4200 rpm for 5 minutes after the shaking.

[0047] As a further improvement of the method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification of the present invention: in step 1: NO2 - Determination of NO3 by N-(1-naphthyl)-amine photometry - Determined by UV spectrophotometry.

[0048] The method of the present invention is used to measure the contribution of ammonia-oxidizing archaea, ammonia-oxidizing bacteria, and complete nitrifying bacteria to soil nitrification, as follows:

[0049] (1) Nitrification potential was used to represent the soil nitrification capacity. 10 g of fresh soil containing oven-dried soil was placed in a 100 ml conical flask. The soil volumetric water content was measured in advance. 100 ml of phosphate buffer (1.5 mM NH4 + and 1mM PO4 3- ) in a conical flask, and then cover the conical flask with a vented lid. Each treatment was repeated three times. The culture flask was placed in a shaker at 180 rpm for 24 h. Six sampling time points (2, 4, 8, 20, 22, and 24 h) were set. 5 ml of the suspension was drawn from the conical flask and placed in a 15 ml centrifuge tube. 5 ml of KCl solution (2 mol L -1 )220rpm shake for 1h, centrifuge at 4200rpm for 5min, then filter the supernatant through a 0.25mm filter membrane to remove the microorganisms and place it in a 4℃ refrigerator to wait for the determination of NO2 - and NO3 - Content. NO2 - Determination of NO3 by N-(1-naphthyl)-amine photometry - Determined by UV spectrophotometry.

[0050] (2) Differentiation of three types of microorganisms: In order to distinguish the contributions of three types of nitrifying functional microorganisms (ammonia oxidizing archaea (AOA), ammonia oxidizing bacteria (AOB) and complete nitrifying bacteria (comammox)), the present invention distinguishes them by improving the multi-inhibitor method. First, NaClO3 (10mM) is added to the buffer to inhibit NO2 - Converted to NO3 - This step is inhibited because AOA and AOB catalyze the first step of the two-step nitrification, so the NO2 in the suspension - The increase is the contribution of AOA+AOB, and at this time the NO3 -The increase is the contribution of the complete nitrifying bacteria comammox. On this basis, adding AOA or AOB specific inhibitors can be achieved through NO2 - The contribution of AOA and AOB was distinguished by the increase in the amount of simvastatin. The present invention selected simvastatin as a specific inhibitor of AOA (12.5 mg g -1 ), DMPP was used as a specific inhibitor of AOB (5% nitrogen content), and the above two inhibitors were added to the buffer solution that had been added with NaClO3. - The contribution of AOA and AOB to soil nitrification can be obtained by measuring the nitrification potential of AOA and AOB. - The nitrification potential represents the nitrification capacity when there is only comammox. In order to distinguish the nitrification capacity of comammox when the three functional genes are metabolized at the same time, the former is named comammoxⅠ and the latter is named comammoxⅡ. - and NO3 - The present invention calculates the nitrification potential of the soil by the following formula:

[0051] Np=R×24×(0.1+V) / m

[0052] Where Np is the soil nitrification potential (mg kg -1 d -1 ), R is the growth rate of NO2-N or NO3-N content (mg L - 1 h -1 ), 0.1 is the volume of buffer solution (L), V is the volume of water in the soil sample (L), and m is the mass of oven-dried soil (kg).

[0053] The method of this invention offers the following advantages over existing technologies: it takes into account interactions between microorganisms; it isolates each microorganism and adds them together, adopting an additive approach that avoids negative values; and it allows for analysis of the ecological niches of microorganisms based on the results. This method can accurately measure the contributions of ammonia-oxidizing archaea, ammonia-oxidizing bacteria, and complete nitrifiers to soil nitrification.

[0054] The present invention sets up a specific method of adding various inhibitors, and sets up a calculation method to distinguish the contributions of various nitrifying microorganisms through two different paths of the nitrification process. The corresponding content in the prior art is to only calculate the contribution of NO3 -To characterize soil nitrification capacity, only one inhibitor is added, without considering the impact of microbial interactions on soil nitrification. Using a subtraction calculation method can easily lead to experimental results that deviate from common sense. Therefore, the present invention can not only measure the relative contributions of AOA, AOB, and comammox to soil nitrification capacity, but also summarize the ecological niches of AOA, AOB, and comammox based on the results.

[0055] Conventional methods prior to this invention first measured the total nitrification capacity of the soil and then subtracted the contribution of two of the three bacteria to obtain the total contribution. However, this method often resulted in negative results in actual measurements because it ignored the interactions between microorganisms. The final calculation in this invention uses an additive algorithm, completely avoiding negative results. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0057] Figure 1 It is a diagram illustrating the specific steps of the present invention.

[0058] Figure 2 is a schematic diagram of the existing method.

[0059] Figure 3 is the nitrification trend of AOA, AOB and comammox within 24 hours;

[0060] Specifically:

[0061] AOA represents the nitrification potential of AOA when both AOB and comammox are inhibited;

[0062] AOB represents the nitrification potential of AOB under the condition that both AOA and comammox are inhibited;

[0063] AOA+AOB represents the combined nitrification potential of AOA and AOB when no inhibition of ammonia-oxidizing microorganisms is applied;

[0064] comammoxⅠ represents the nitrification potential of comammox when ammonia-oxidizing microorganisms are not inhibited;

[0065] comammoxⅡ represents the nitrification potential of comammox when both AOA and AOB are inhibited;

[0066] In each small picture: ●G, ■U, ▲N.

[0067] Figure 4 Comparison of the nitrification contribution percentages of AOA, AOB, and comammox, as well as their nitrification potential. DETAILED DESCRIPTION

[0068] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0069] Example 1: The relative contributions of AOA, AOB, and comammox to nitrification in the incubated soil were determined by sequentially performing the following steps:

[0070] 1) Soil culture experiment:

[0071] The culture soil comes from Guyuan City, Ningxia Hui Autonomous Region, which has a temperate continental climate. The average annual temperature is 8.5℃, and the average annual precipitation is 450mm. The soil in the study area is gray-calcium soil with a coarse texture and is sandy loam. Three treatments were set up: CK (control, no winter irrigation), WI1 (1 year of organic liquid fertilizer winter irrigation), and WI2 (two consecutive years of organic liquid fertilizer winter irrigation). Each treatment had 3 replicates, a total of 9 plots, and each plot had an area of ​​1 mu. WI1 was winter irrigated with PW in October 2018, with a winter irrigation volume of 80m 3 ha -1 WI2 carried out winter irrigation of PW in October 2018 and October 2019, with a winter irrigation volume of 80m 3 ha -1 After irrigation was completed, potatoes were planted in May of the following year. Soil was collected on November 13, 2020, and five topsoil samples (0-20 cm) were collected from each plot and mixed.

[0072] Three types of exogenous additives were added to the above three treatment soils, namely glycine (N-100 mg kg -1 ), urea (N-100mg kg -1 ), glucose (C-200mg kg -1 )+ammonium chloride (N-100mg kg -1 ), and then the corresponding 9 experimental groups were subjected to pre-incubation treatment for nitrification reaction of the soil (optional), specifically as follows: after adding exogenous additives to the soil of the above 9 experimental groups according to the treatment, they were incubated in a closed manner at 25°C and a soil moisture content of 60% of the field water holding capacity for 30 days, and high-purity air was ventilated once every 7 days; after the incubation, the soil was taken as the test soil for subsequent experiments.

[0073] Table 1 Experimental treatment designs

[0074]

[0075] 2) Determination of soil nitrification potential after cultivation

[0076] After collecting the soil after each treatment and cultivation, the nitrification contribution rate of AOA, AOB and comammox in each treatment group of the soil to be tested was measured according to the multi-inhibition measurement method of the present invention.

[0077] Specifically, for any of the treatment groups described in Table 1 above, perform the following steps:

[0078] (1) Setting the determination method of nitrification potential:

[0079] Nitrification potential is used to represent the soil nitrification capacity. The volumetric water content of the soil to be tested is measured in advance. 10g of the test soil containing dried soil is placed in a 100ml conical flask, and 100ml of phosphate buffer (1.5mM NH4 + and 1mM PO4 3- ) and the corresponding inhibitor were prepared into a soil suspension to be tested in a conical flask, which was then covered with a vented lid. Each treatment was repeated three times. The culture flask was placed in a shaker at 180 rpm and 25°C for 24 h. Six sampling time points (2, 4, 8, 20, 22, and 24 h) were set. 5 ml of the suspension was drawn from the conical flask and placed in a 15 ml centrifuge tube. 5 ml of KCl solution (2 mol L -1 )220rpm shake for 1h, centrifuge at 4200rpm for 5min, filter the supernatant through a 0.25mm filter membrane to remove the microorganisms, and place it in a 4℃ refrigerator. The samples taken at each time point need to be NO2 - and NO3 - Determination of NO2 content. - Determination of NO3 by N-(1-naphthyl)-amine photometry - Determined by UV spectrophotometry.

[0080] Note: Oven-dried soil is dried at 105°C to a constant weight. The volume of water in the soil sample is calculated based on the change in weight before and after drying. This is a standard technique.

[0081] (2) Differentiation of three types of microorganisms: In order to distinguish the contributions of three types of nitrifying functional microorganisms (ammonia oxidizing archaea (AOA), ammonia oxidizing bacteria (AOB) and complete nitrifying bacteria (comammox)), the multi-inhibitor method was improved to distinguish them.

[0082] First, NaClO3 (10 mM) was added to a soil suspension consisting of 10 g of fresh soil and 100 ml of phosphate buffer to prepare the soil suspension to be tested, named soil suspension I, to measure NO2 - Converted to NO3 -This step is inhibited because AOA and AOB catalyze the first step of the two-step nitrification, so the NO2 in the suspension - The increase is the contribution of AOA+AOB, and at this time, the NO3 - The increase is due to the contribution of complete nitrifying bacteria comammox.

[0083] On the basis of the above, adding AOA or AOB specific inhibitors can - The contributions of AOA and AOB were distinguished by the increase in soil concentration. Simvastatin was selected as the specific inhibitor of AOA (12.5 mg of simvastatin was added per g of soil), and DMPP was selected as the specific inhibitor of AOB (5% nitrogen content, that is, because the nitrogen content in the buffer was 1.5 mM NH4 + , so the concentration of DMPP should be 0.075mM), the above two inhibitors were added to the soil suspension that had been added with NaClO3, and the NO2 - The contribution of AOA and AOB to soil nitrification can be obtained by calculating the nitrification potential of soil nitrification.

[0084] If these three inhibitors are added to the soil suspension at the same time, and then the NO3 - The obtained nitrification potential represents the nitrification ability when there is only comammox. In order to distinguish the nitrification ability of comammox when the three functional genes are metabolized simultaneously, the present invention named the former (i.e., the nitrification potential of comammox when AOA, AOB and comammox are not inhibited) as comammoxⅠ, and the latter (i.e., the nitrification potential of comammox when both AOA and AOB are inhibited) as comammoxⅡ. After measuring NO2 - and NO3 - After determining the content of soil nitrification potential, the formula was used to calculate the soil nitrification potential.

[0085] Specifically,

[0086] To the test soil containing 10 g of oven-dried soil, 100 ml of phosphate buffer was added, and then NaClO₃ was added to a final concentration of 10 mM. The soil suspension with NaClO₃ added was named soil suspension I.

[0087] The soil suspension I was measured according to the method of step 1 at six sampling time points. - Content, obtain NO2 - The linear regression equation corresponding to the concentration and sampling time is used to obtain the slope R AOA+AOB ;

[0088] The soil suspension I was measured according to the method of step 1 at six sampling time points. - Content, obtain NO3 - The linear regression equation corresponding to the concentration and sampling time is used to obtain the slope R comammoxⅠ ;

[0089] Simvastatin was added to soil suspension I at a dosage of 12.5 mg of simvastatin per g of soil, and the resulting suspension was named simvastatin soil suspension;

[0090] The simvastatin soil suspension was measured according to the method of step 1 to determine the NO2 - Content, obtain NO2 - The linear regression equation corresponding to the concentration and sampling time is used to obtain the slope R AOB ;

[0091] DMPP was added to the soil suspension I until the concentration of DMPP was 0.075 mM; the result was named DMPP soil suspension;

[0092] The DMPP soil suspension was measured according to the method in step 1 to determine the NO2 at six sampling time points. - Content, obtain NO2 - The linear regression equation corresponding to the concentration and sampling time is used to obtain the slope R AOA ;

[0093] Simvastatin and DMPP were added to soil suspension I simultaneously until the DMPP concentration reached 0.075 mM, with 12.5 mg of simvastatin added per g of soil; the resulting suspension was designated as simvastatin + DMPP soil suspension.

[0094] Simvastatin + DMPP soil suspension was measured according to the method of step 1 at six sampling time points. - Content, get NO3 - The linear regression equation corresponding to the concentration and sampling time is used to obtain the slope R comammoxⅡ .

[0095] The formula for calculating the nitrification potential of soil is:

[0096] Np=R×24×(0.1+V) / m

[0097] Where Np is the soil nitrification potential (mg kg -1 d -1 ), R is the growth rate of NO2-N or NO3-N content (mg L - 1 h -1), 0.1 is the volume of buffer solution (L), V is the volume of water in the soil sample (L), and m is the mass of oven-dried soil (kg).

[0098] Take "CK-Glycine" as an example for detailed explanation:

[0099] The soil was supplemented with glycine (N-100 mg kg -1 ) and cultivate the soil for 30 days as the test soil.

[0100] 1) Add NaClO3 (10 mM) to a soil suspension consisting of 10 g of fresh soil and 100 ml of phosphate buffer to reduce NO2 - Converted to NO3 - This step is to suppress the NO2 in the samples at six sampling time points. - The contents are 0.14627, 0.15801, 0.26363, 0.89738, 1.04994, and 1.09689, respectively. The above data are subjected to linear regression to obtain the slope R AOA+AOB is 0.04509 (the slope is the slope of AOA+AOB); that is, the linear regression equation is Figure 3 -y=0.01+0.05x in CK-AOA+AOB-G, where X represents the sampling time and Y represents the NO2 in the collected sample - concentration.

[0101] Determination of NO3 in samples at six sampling time points - The contents are 2.7194048, 2.669632, 2.3709952, 3.16736, 3.2216576, and 3.3076288, respectively. The above data are subjected to linear regression to obtain the slope R comammoxⅠ is 0.03662 (the slope is the slope of comammox I). That is, the linear regression equation is Figure 3 -y=2.72+0.04x in CK-comammoxⅠ-G, where X represents the sampling time and Y represents the NO3 in the collected sample. - concentration.

[0102] 2) Add NaClO3 (10 mM) and simvastatin (12.5 mg of simvastatin per 1 g of soil, 125 mg of simvastatin per 10 g of soil) to a soil suspension consisting of 100 ml of phosphate buffer. - Converted to NO3 - This step is to inhibit and specifically inhibit AOA. At this time, the NO2 in the samples at six sampling time points is measured.- The contents are 0.0055532, 0.0067268, 0.0125948, 0.0483896, 0.0571916, and 0.0612992, respectively. The above data are subjected to linear regression to obtain the slope R AOB is 0.05107 (the slope is the slope of AOB); that is, the linear regression equation is Figure 3 -y in CK-AOB-G = -0.03 + 0.05x, where X represents the sampling time and Y represents the NO2 in the collected sample - concentration.

[0103] NaClO3 (10 mM) and DMPP (5% nitrogen content, i.e., 1.5 mM NH4 in the buffer solution) were added to a soil suspension consisting of 10 g of fresh soil (dried soil) and 100 ml of phosphate buffer. + , so the concentration of DMPP should be 0.075mM) for NO2 - Converted to NO3 - This step is to inhibit and specifically inhibit AOB. At this time, the NO2 in the samples at six sampling time points is measured. - The contents are 0.0026192, 0.0037928, 0.0043796, 0.0084872, 0.00614, and 0.0084872, respectively. The above data are subjected to linear regression to obtain the slope R AOA is 0.00564 (the slope is the slope of AOA). That is, the linear regression equation is Figure 3 -y in CK-AOA-G = 0.04 + 0.01x, where X represents the sampling time and Y represents the NO2 in the collected sample - concentration.

[0104] 3) NaClO3 (10 mM), simvastatin (12.5 mg of simvastatin per g of soil) and DMPP (5% nitrogen content, i.e., the nitrogen content in the buffer solution is 1.5 mM NH4) were added to a soil suspension consisting of 10 g of fresh soil from oven-dried soil and 100 ml of phosphate buffer. + , so the concentration of DMPP should be 0.075mM), for NO2 - Converted to NO3 - This step inhibits and specifically inhibits AOA and AOB. Determine the NO3 in the samples at six sampling time points - The contents are 1.1402496, 1.9547136, 2.1583296, 2.1990528, 2.1447552, and 2.33431, respectively. The above data are subjected to linear regression to obtain the slope R comammoxⅡis 0.03241 (the slope is the slope of comammox II). That is, the linear regression equation is Figure 3 -y=3.65+0.03x in CK-comammoxⅡ-G, where X represents the sampling time and Y represents the NO3 in the collected sample. - concentration.

[0105] Substitute the above data into the formula Np=R×24×(0.1+V) / m, m=10g, V=0.0007L, and the final calculation formula is Np=R×241.68. So Np AOA+AOB =R AOA+AOB ×241.68=10.8973512,Np comammoxⅠ =R comammoxⅠ ×241.68=8.8503216,Np AOA =R AOA ×241.68=1.3536,Np AOB =R AOB ×241.68=12.3426,Np comammoxⅡ =R comammoxⅡ × 241.68 = 7.83284. The Np obtained for each treatment group in Table 1 above is as follows Figure 4 shown, and Figure 4 The results were integrated and the percentages of various microorganisms were calculated, which was Figure 4 .

[0106] The results showed that soil nitrification was mainly driven by the nitrification capacity of AOB and comammox (≥85%), while AOA contributed less than 15% to soil nitrification. Furthermore, the nitrification potential of AOA and AOB (AOA+AOB) was lower than the sum of the nitrification potentials of AOA and AOB individually. This suggests that there may be a competitive relationship between AOA and AOB. Because of this competitive relationship, neither AOA nor AOB can fully exert their nitrification capacity, resulting in the sum of the nitrification potentials of AOA and AOB being higher than the nitrification potentials of both. At the same time, the nitrification capacity and proportion of comammox II in the total nitrification potential were always lower than those of comammox I in all treatments. This indicates that when comammox, AOA, and AOB jointly nitrify, the nitrification function of comammox is promoted. To summarize the above results, AOB and comammox play the main roles in soil nitrification, and there is a competitive relationship between AOA and AOB, and a mutually beneficial relationship between AOA and comammox, as well as between AOB and comammox.

[0107] Comparative treatment of CK-Glycine, the calculation method of the present invention is changed to Np as described in the prior art AOA =Np AOA+AOB -Np AOB The remaining results are similar to those in Example 1 above. The results are: AOB = 12.3426; AOA = -1.4452488, with the AOA result being a negative number. Therefore, compared to Example 1, the improved algorithm in this invention, which takes into account the effects of microbial interactions, avoids negative results. Furthermore, the results can be used to summarize the ecological niches of AOA, AOB, and comammox.

[0108] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. A method for measuring the contribution of ammonia-oxidizing microorganisms to soil nitrification, characterized in that The following steps are involved: Step 1: Set the method for measuring nitrification potential: The soil to be tested was prepared into a soil suspension and incubated on a shaking table for 24 h; Set six sampling time points; At each sampling time, the following operations were performed: 5 ml of the incubated soil suspension was taken, mixed with 5 ml of KCl solution, shaken, and then centrifuged. The supernatant was filtered through a 0.25 mm filter membrane and the NO2 - and NO3 - content; Step 2: Differentiation of three types of microorganisms: 1) Add 100 ml of phosphate buffer to 10 g of the soil to be tested and then add NaClO3 to a final concentration of 10 mM; the soil suspension with NaClO3 added is named soil suspension I; The soil suspension I was measured according to the method of step 1 to measure NO2 at six sampling time points. - Content, obtain NO2 - The linear regression equation corresponding to the concentration and sampling time is used to obtain the slope R AOA+AOB ; The soil suspension I was measured according to the method of step 1 at six sampling time points. - Content, obtain NO3 - The linear regression equation corresponding to the concentration and sampling time is used to obtain the slope R comammoxⅠ ; 2) It includes the following two steps: 2.1) Simvastatin was added to soil suspension I at a ratio of 12.5 mg of simvastatin per g of soil, and the resulting suspension was named simvastatin soil suspension; The simvastatin soil suspension was measured according to the method of step 1 to determine the NO2 - Content, obtain NO2 - The linear regression equation corresponding to the concentration and sampling time is used to obtain the slope R AOB ; 2.2) Add DMPP to the soil suspension I until the concentration of DMPP is 0.075 mM; the resulting suspension is named DMPP soil suspension; The DMPP soil suspension was measured according to the method in step 1 to determine the NO2 at six sampling time points. - Content, obtain NO2 - The linear regression equation corresponding to the concentration and sampling time is used to obtain the slope R AOA ; 3) Simvastatin and DMPP were added to soil suspension I simultaneously until the DMPP concentration reached 0.075 mM, with 12.5 mg of simvastatin added per g of soil. The resulting suspension was designated as simvastatin + DMPP soil suspension. Simvastatin + DMPP soil suspension was measured according to the method of step 1 at six sampling time points. - Content, get NO3 - The linear regression equation corresponding to the concentration and sampling time is used to obtain the slope R comammoxⅡ ; Step 3: Calculate the nitrification potential of the soil.

2. The method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification according to claim 1, characterized in that: The six sampling time points set in step 1 correspond to the 2nd, 4th, 8th, 20th, 22nd, and 24th hours of culture, respectively.

3. The method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification according to claim 2, characterized in that: Np=R×24×(0.1+V) / m; Np is the soil nitrification potential, the unit is mg kg -1 d -1 ; R is the growth rate of NO2-N or NO3-N content, the unit is mgL -1 h -1 ; 0.1 is the volume of phosphate buffer, unit is L; V is the volume of water in the soil sample, unit is L; m is the mass of oven-dried soil, unit is kg.

4. The method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification according to claim 3, characterized in that: In phosphate buffer, NH4 + The concentration is 1.5mM, PO4 3- The concentration is 1 mM.

5. The method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification according to claim 4, characterized in that: The KCl concentration in KCl solution is 2 mol L -1 .

6. The method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification according to any one of claims 3 to 5, characterized in that: When m = 10 g, V = 0.0007 L, Np = R × 241.68; Np AOA+AOB =R AOA+AOB ×241.68; Np comammoxⅠ =R comammoxⅠ ×241.68; Np AOA =R AOA ×241.68; Np AOB =R AOB ×241.68; Np comammoxⅡ =R comammoxⅡ ×241.68。 7. The method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification according to claim 6, characterized in that In the step 1: The mixture was shaken at 220 rpm for 1 h and then centrifuged at 4200 rpm for 5 min.

8. The method for measuring the contribution of ammonia oxidizing microorganisms to soil nitrification according to claim 7, characterized in that In the step 1: NO2 - Determination of NO3 by N-(1-naphthyl)-amine photometry - Determined by UV spectrophotometry.

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