A method for synchronous determination of soil microbial carbon and nitrogen content

Abstract

The present invention provides a method for synchronous determination of soil microbial carbon and nitrogen content, which can accurately and quickly evaluate soil microbial activity and its role in carbon and nitrogen cycle. The specific steps include: firstly, fresh soil samples passing through a 2 mm sieve are divided into a control group and a treatment group, and 0.5 mol L⁻¹ potassium sulfate solution is added to each group, and ethanol-free chloroform is also added to the treatment group; oscillate at a frequency of 180 r min⁻¹ at 25°C; then centrifuge the mixture at a speed of 3500 r min⁻¹ for 10 minutes; oxygen is introduced into the supernatant after centrifugation in a double-mouth reactor for 30 minutes to enhance the reaction; finally, the reaction liquid is placed in a TOC element analyzer for sample analysis, and the carbon and nitrogen content is detected at the same time. By calculating the difference between the carbon and nitrogen content of the treatment group and the control group and dividing it by the conversion coefficient, the soil microbial biomass carbon and nitrogen can be obtained. The present invention realizes the synchronous determination of soil microbial carbon and nitrogen content, is easy to operate, and is suitable for the evaluation of soil health and soil quality.

Description

Technical Field

[0001] The present invention relates to the field of analytical detection technology (G01N), and in particular to a method for synchronously determining the carbon and nitrogen contents of soil microorganisms. Background Art

[0002] Soil microbial biomass is one of the important indicators for evaluating soil fertility and health. Microbial biomass carbon and nitrogen, as the most important components of soil microbial biomass, can not only reflect the biological quality of soil, but also play an important role in soil material conversion, energy flow and biogeochemical cycle. The determination methods of soil microbial biomass carbon and nitrogen include fumigation extraction method (CN 101419226B, CN118010451A), microwave extraction method (CN 109085322 A), microbial DNA conversion method (CN 109916840 A), etc.

[0003] The fumigation extraction method is currently the most widely used and recognized standard method for determining soil microbial biomass carbon and nitrogen. Its principle is to vacuum the dryer containing soil samples and chloroform, so that the chloroform boils under vacuum and becomes chloroform vapor that diffuses in the soil pores to destroy the cell membrane structure of soil microorganisms and promote the release of microbial cell active substances, and then use K2SO4 solution to extract soil carbon and nitrogen. This method includes two main steps: gaseous chlorine simulated air fumigation and K2SO4 extraction. In order to ensure the maximum release of microbial cell active substances, a fumigation time of more than 24 hours is usually required, and the extraction process is about 30 minutes. However, this method has problems such as poor sealing leading to fumigation failure, unsatisfactory fumigation effect on heavy or high water content soil, and large amount of soil samples.

[0004] The principle of microwave extraction is to use 400-550W microwaves to kill soil microorganisms, promote the release of active substances in microbial cells, and extract soil nitrogen with K2SO4 solution. This method includes two main steps: microwave treatment and K2SO4 extraction, which greatly shortens the processing time. However, this method is only suitable for the determination of microbial biomass nitrogen, and it is not certain whether it can be used for the determination of microbial biomass carbon. In addition, the determination of microbial biomass carbon and nitrogen mostly uses fresh soil, and the microwave thermal effect will increase the soil temperature, which is easy to cause problems such as loss of microbial active substances or protein denaturation, affecting the determination results.

[0005] The principle of the microbial DNA conversion method is that there is a significant positive correlation between the soil microbial DNA content and the soil microbial biomass carbon and nitrogen. Based on the sample microbial DNA content and the soil microbial biomass carbon and nitrogen conversion coefficient (carbon is 37.20, nitrogen is 4.02), the microbial biomass carbon and nitrogen are calculated by the microbial DNA content. This method includes the extraction of soil microbial DNA, the determination of DNA concentration, the determination of the microbial biomass carbon and nitrogen conversion coefficient in a specific microhabitat, and the calculation of microbial biomass carbon and nitrogen. However, this method requires high DNA extraction technology, and the DNA extraction reagent package used is expensive and the detection cost is high.

[0006] In view of these limitations of the prior art, the present invention proposes a simple and rapid method for synchronously determining the carbon and nitrogen content of soil microorganisms, aiming to solve the above problems. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a method for synchronously determining the carbon and nitrogen content of soil microorganisms, and the steps of the determination method are as follows:

[0008] (1) Weigh two groups of fresh soil that have passed through a 2 mm sieve and place them in 100 mL centrifuge tubes, which are respectively named the control group and the treatment group;

[0009] (2) Add 0.5 mol L -1 The potassium sulfate solution was added to the centrifuge tubes of the treatment groups in turn. -1 of potassium sulfate solution and ethanol-free chloroform;

[0010] (3) The mixture from step (2) was placed at 25°C and heated at 180 r min -1 The frequency oscillation of

[0011] (4) Place the mixture from step (3) in a centrifuge at 3500 r min -1 Centrifuge for 10 min at a frequency of

[0012] (5) The supernatant from step (4) is transferred into a double-mouthed reactor, one end of which is connected to an air duct and the other end is an exhaust port; in a light-proof condition, O2 is introduced into the supernatant through the air duct, and the exhaust gas is discharged in the form of bubbling, and the exhaust gas passes through a high-temperature catalyst for 30 minutes; wherein the chloroform-containing liquid reacts chemically with O2 as follows:

[0013] 2CHCl3+2H2O+O2→2CO2+6HCl

[0014] (6) After the reaction in step (5) is completed, the liquid is transferred to a sample bottle and placed in the injection port of a TOC element analyzer to analyze the carbon and nitrogen content at the same time. The difference in carbon and nitrogen content between the treatment group and the control group multiplied by the conversion coefficient is the soil microbial biomass carbon and nitrogen.

[0015] Wherein, the chloroform is directly added to the soil-potassium sulfate mixture.

[0016] As an embodiment of the present invention, the volume of chloroform added and the mass of the soil sample are 50 μL:1g, 100 μL:1g and 200 μL:1g, and the optimal ratio is 100 μL:1g.

[0017] As an embodiment of the present invention, the oscillation time is 1-4 h, optimally 1 h.

[0018] As an embodiment of the present invention, the oxygen introduction time is 30 min.

[0019] The second aspect of the present invention provides a method for synchronously determining the carbon and nitrogen content of soil microorganisms, which is applied to the field of agricultural detection.

[0020] Beneficial effects: By adopting the above technical solution, the present invention has the following beneficial effects:

[0021] (1) The present invention proposes for the first time to use O2 to remove residual chloroform in the soil microbial biomass carbon and nitrogen extraction system. The residual chloroform can be converted into CO2 by a chemical reaction with O2 and discharged. While removing the residual chloroform in the sample, the pollution and risk of chloroform to the environment is reduced.

[0022] (2) The present invention is the first to directly add liquid chloroform to the mixed system of K2SO4 and soil and then oscillate to extract carbon and nitrogen from soil microbial biomass. Chloroform and soil can fully contact, stir and mix, which has a better killing effect on soil microorganisms. It can effectively eliminate the problem of inaccurate measurement results caused by poor sealing, heavy soil, high water content, etc., and the processing efficiency of each sample processed by the present invention is improved by 93.2% compared with the traditional method. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the process of the present invention;

[0024] Figure 2 It is a schematic diagram showing the consistency of the results of the method of the present invention and the traditional method (biomass carbon);

[0025] Figure 3 Schematic diagram of the consistency of the results of the method of the present invention and the traditional method (biomass nitrogen)

[0026] Figure 4 Schematic diagram showing the stability comparison between the method of the present invention and the traditional method. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] A method for synchronously determining the carbon and nitrogen content of soil microorganisms comprises: preparing 0.5 mol L -1 Weigh a fresh soil sample and put it into a centrifuge tube. First add potassium sulfate solution at a liquid-to-soil ratio of 4:1, and then add ethanol-free chloroform at an appropriate proportion. Oscillate at 25°C. After centrifugation, pass O2 into the supernatant under light-proof conditions for a period of time, then transfer it to a sample bottle, and use a TOC elemental analyzer to analyze the carbon and nitrogen content of the supernatant.

[0029] The ethanol-free chloroform was directly added to the mixed solution of soil and potassium sulfate, the ratio of soil sample mass to chloroform was 1 g:50 μL, 1 g:100 μL and 1 g:200 μL, the oscillation time was 1, 2, and 4 h, and the O2 introduction time was 30 min.

[0030] The present invention is further explained below in conjunction with specific implementation modes.

[0031] Examples 1-4

[0032] like Figure 1 As shown, the method of the present invention is to prepare 0.5 mol L -1 The potassium sulfate solution was added to the fresh soil samples and placed in two sets of centrifuge tubes. Group A was added with potassium sulfate solution at a liquid-to-soil ratio of 4:1; Group B was first added with potassium sulfate solution at a liquid-to-soil ratio of 4:1, and then ethanol-free chloroform was added at a ratio of 1 g:50 μL, 1 g:100 μL, and 1 g:200 μL. The two groups of mixtures were placed at 25°C and shaken for 1, 2, and 4 h, and then centrifuged (3500 r min -1 The samples were centrifuged at a frequency of 10 min under light-proof conditions, and O2 was introduced into the supernatant for 30 min. The exhaust gas passed through a high-temperature catalyst, and then the supernatant was transferred to a sample bottle. The carbon and nitrogen contents were determined using a TOC elemental analyzer. The difference in carbon and nitrogen contents between group A and group B samples divided by the conversion coefficient was the microbial biomass carbon and nitrogen.

[0033] Traditional method: Weigh fresh soil samples and place them in two sets of small beakers and place them in a vacuum glass desiccator. In Group A, place 60 mL of ethanol-free chloroform (with a small amount of zeolite) and 30 mL of dilute sodium hydroxide solution, and evacuate the chloroform to boil and evaporate and diffuse into the soil pores. After 24 hours, evacuate again to make the chloroform in the soil pores and the chloroform in the desiccator become gaseous and evaporate and discharge to the external environment. Then take out the small beaker and add 0.5 mol L -1 30 mL of dilute sodium hydroxide solution was placed in group B without chloroform. After 24 h, the beaker was taken out and 0.5 mol L -1 of potassium sulfate solution. The two groups of beakers were then placed at 25°C and shaken for 30 min. After filtering, the two groups of filtrates were transferred to sample bottles. The carbon and nitrogen contents were determined by TOC element analyzer. The difference in carbon and nitrogen contents between group A and group B samples divided by the conversion coefficient was the microbial biomass carbon and nitrogen. Based on the optimized parameters, six typical farmland red soil samples with different microbial biomass carbon and nitrogen (provided by the Qiyang Red Soil Experimental Station of the Chinese Academy of Agricultural Sciences) were selected for experiments, and the soil microbial biomass carbon and nitrogen were determined by the method of the present invention and the traditional method, respectively.

[0034] At the addition ratio of 1 g:0.50 μL and 1 g:100 μL, the carbon and nitrogen of microbial biomass measured after 1 h, 2 h and 4 h of oscillation were slightly different from those of the traditional method; while the addition ratio of 1 g:200 μL resulted in higher carbon and nitrogen of microbial biomass than those of the traditional method, which may be related to the residual effect caused by excessive addition of chloroform. Therefore, the optimal parameters can be set to 1 g:100 μL and 1 h of oscillation.

[0035] Table 1 Effect of different chloroform dosage on the determination results after 1 h of oscillation

[0036]

[0037] Table 2 Effect of different chloroform dosage on the determination results after 2 h of oscillation

[0038]

[0039] Table 3 Effect of different chloroform dosage on the determination results after 4 h of oscillation

[0040]

[0041] Compared with the traditional method, there is basically no significant difference in the carbon and nitrogen of microbial biomass determined by the method of the present invention (Table 4). The results of linear regression analysis show that ( Figure 2 , Figure 3), the soil microbial biomass carbon and nitrogen determined by the method of the present invention have a very significant correlation with the results of the traditional method ( P <0.01, R ≥0.86), indicating that the carbon and nitrogen content of microbial biomass determined by the method of the present invention is in good agreement with the results determined by the traditional method.

[0042] By calculating the mean and overall standard deviation of the samples, it can be seen that the method of the present invention has better stability than the traditional method (Table 5, Figure 4 ); In Table 5, the absolute value of the test value deviation is divided by the test value. For example, the test value deviation of the traditional method of red soil one is 2.76 mg, and the test value is 58.21 mg. The calculated deviation ratio is 0.0474. The same is true for other data. Then, the deviation ratios of red soil one to red soil five are used as samples to obtain the sample mean and population standard deviation. The smaller the value, the better. A small sample mean indicates a small deviation ratio, and a small population standard deviation indicates better result stability. Figure 4 It can be seen that the deviation and stability of the method of the present invention are better than those of the traditional method.

[0043] The present invention directly adds liquid chloroform to the soil and then shakes it, so that the chloroform and the soil can fully contact, stir and mix, and the killing effect on soil microorganisms is better, which significantly improves the treatment efficiency by 93.2% (Table 6).

[0044] Table 4 Differences between the results of the method of the present invention and the traditional method

[0045]

[0046] Table 5 Comparison of the stability of the results of the method of the present invention and the traditional method

[0047]

[0048] Table 6 Time cost calculation

[0049]

[0050] Note: The traditional method is to fumigate first and then extract, and the time cost is calculated step by step; the method of the present invention combines the fumigation and extraction steps, and the time cost is calculated together; the supernatant preparation step of the traditional method is only centrifugation for 0.17 h, while the supernatant preparation step of the method of the present invention is to centrifuge for 0.17 h and then introduce O2 for 0.5 h.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for synchronously determining the carbon and nitrogen content of soil microorganisms, characterized in that: The steps of the assay are as follows: S1. Weigh two groups of fresh soil that have passed through a 2 mm sieve and place them in 100 mL centrifuge tubes, which are recorded as the control group and the treatment group respectively; S2, adding 0.5 mol·L-1 potassium sulfate solution to the centrifuge tube of the control group in step S1, and adding 0.5 mol·L-1 potassium sulfate solution and ethanol-free chloroform to the centrifuge tube of the treatment group in sequence; The ratio of soil to chloroform was 1 g:100 μL; S3, the mixture of step S2 was placed at 25°C and heated at 180 r·min -1 The frequency oscillation of S4, place the mixture in step S3 in a low-speed centrifuge at 3500 r·min -1 Centrifuge for 10 min at a frequency of S5, transferring the supernatant from step S4 into a double-port reactor, one end of which is connected to an air duct and the other end is an exhaust port; in a light-proof condition, O2 is introduced into the supernatant through the air duct, and the gas is discharged in the form of bubbling, and the exhaust gas passes through a high-temperature catalyst; S6. After the reaction in step S5 is completed, the liquid is transferred to a sample bottle, placed in the injection port of a TOC element analyzer, and the carbon and nitrogen contents are analyzed simultaneously; the difference in carbon and nitrogen contents between the treatment group and the control group is multiplied by the conversion coefficient to obtain the soil microbial biomass carbon and nitrogen.

2. The method for synchronous determination of soil microbial carbon and nitrogen content according to claim 1, characterized in that: In step S2, chloroform is directly added to the soil-potassium sulfate mixture.

3. The method for synchronous determination of soil microbial carbon and nitrogen content according to claim 1, characterized in that: In step S3, the oscillation time is 1 to 4 h.

4. The method for synchronous determination of soil microbial carbon and nitrogen content according to claim 1, characterized in that: In step S3, the oscillation time is 1 h.

5. The method for synchronous determination of soil microbial carbon and nitrogen content according to claim 1, characterized in that: In step S5, the oxygen is introduced for 0.5 h.

6. Application of the method for synchronous determination of soil microbial carbon and nitrogen content according to any one of claims 1 to 5 in the field of agricultural testing.

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