CN112379078A - Method for measuring carbon isotope content of soil microorganisms - Google Patents

Method for measuring carbon isotope content of soil microorganisms Download PDF

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CN112379078A
CN112379078A CN202011137427.8A CN202011137427A CN112379078A CN 112379078 A CN112379078 A CN 112379078A CN 202011137427 A CN202011137427 A CN 202011137427A CN 112379078 A CN112379078 A CN 112379078A
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蒋新宇
程炯
刘平
刘晓南
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Institute of Eco Environmental and Soil Sciences of Guangdong Academy of Sciens
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Abstract

The invention discloses a method for measuring carbon isotope content of soil microorganisms, which comprises the following steps: dividing the soil samples into two groups, wherein the first group of samples are fumigated by chloroform and then extracted by potassium sulfate solution to obtain filtrate A, and the second group of samples are directly extracted by the potassium sulfate solution without being fumigated to obtain filtrate B; measuring the total carbon content of filtrate A and filtrate B, sealing the rest part, freeze drying to obtain powder A and powder B, and respectively processing powder A and powder B13C content determination and calculation of soil sample microorganisms13And (4) C content. The method is simple and convenient, can improve the content of carbon isotopes in a unit volume sample, and can detect low concentrationThe sample has high sensitivity, the lowest concentration of the lowest detectable sample is 0.5mg/L, and the cost is lower.

Description

Method for measuring carbon isotope content of soil microorganisms
Technical Field
The invention belongs to the technical field of soil microbial carbon determination, and particularly relates to a method for determining the carbon isotope content of soil microbes.
Background
In global change studies, carbon cycling is at a central position. In the past 200 years, due toThe increasing concentration of atmospheric CO2, carbon recycling and greenhouse gas issues are becoming more and more important. The focus of global carbon cycle research is to ascertain carbon source, carbon sink, and to quantify. The soil carbon cycle is an important ring of global carbon cycle, the global average thickness of the soil is only 3m, but the carbon content in the soil is more than 2 times of the atmospheric carbon content. Therefore, changes in soil carbon content can contribute to atmospheric CO2Concentration and global carbon cycle contribute to significant impact. Soil microorganisms play an important role in regulating and controlling the circulation of soil elements, and a large amount of exogenous organic carbon enters the soil environment through the action of microorganisms or is retained in the soil environment for a long time in the form of microbial residues.
In the field of soil carbon cycle research, the environmental behavior of soil carbon is traced and researched by means of an isotope tracing technology, and the soil carbon is quantified in different carbon libraries, which is the key point of relevant research. Among them, the carbon isotope assay of soil microorganisms requires the help of high-precision instruments, the technology is complex, and the cost of single assay is expensive. The carbon extraction of soil microorganism is mainly carried out by chloroform fumigation-K2SO4The extraction is realized, and because the liquid sample is obtained after extraction, the sample introduction concentration often cannot meet the requirement, so that the detection result has large error, and even the result cannot be detected. Meanwhile, the isotope determination cost for the liquid is high, and the single-sample test cost is usually about 1000 yuan, which greatly limits the analysis and determination of the carbon isotope of the soil microorganism.
Disclosure of Invention
The invention aims to provide a method for measuring the carbon isotope content of soil microorganisms.
The technical scheme adopted by the invention is as follows:
the invention provides a method for measuring carbon isotope content of soil microorganisms, which comprises the following steps:
s1: dividing the soil samples into two groups, wherein the first group of samples are fumigated by chloroform and then extracted by potassium sulfate solution to obtain filtrate A, and the second group of samples are directly extracted by the potassium sulfate solution without being fumigated to obtain filtrate B;
s2: measuring the total carbon content of filtrate A and filtrate B, respectively sealing the rest part, freeze drying to obtain powder A and powder B, and freeze preserving;
s3: respectively carrying out the steps of13C content is measured, and then the carbon isotope content of the soil microorganisms is calculated.
According to the method disclosed by the invention, the specific method for calculating the carbon isotope content of the soil microorganisms in the step S3 comprises the following steps: by passing
Figure BDA0002737177900000011
Calculating microorganisms of soil samples13C content;
wherein the content of the first and second substances,13C-MBC is the carbon isotope content, delta, of soil microorganisms13Cf and delta13Cnf for powder A and powder B respectively13C abundance, Cf and Cnf are the total carbon content of filtrate a and filtrate B, respectively.
According to the method of the invention, the soil sample obtained in step S1 is sieved by a 4mm sieve.
According to the method, the chloroform fumigation treatment in the step S1 is specifically to place the sample in a chloroform environment for closed culture for 20-28 h.
Preferably, according to the method of the present invention, the chloroform fumigation treatment in step S1 is specifically to place the sample in a chloroform environment for sealed culture for 24 hours.
According to the method, the concentration of the potassium sulfate in the step S1 is 0.4-0.6 mol/L.
Preferably, according to the method of the present invention, the potassium sulfate concentration in step S1 is 0.5 mol/L.
According to the method, the soil-water ratio of the first group of samples to the potassium sulfate solution in the step S1 is 1 (4-6).
Preferably, the soil-to-water ratio of the first set of samples to the potassium sulfate solution in step S1 is 1:5 according to the method of the present invention.
According to the method, the soil-water ratio of the second group of samples to the potassium sulfate solution in the step S1 is 1 (4-6).
Preferably, the soil-to-water ratio of the second set of samples to the potassium sulfate solution in step S1 is 1:5 according to the method of the present invention.
According to the method of the present invention, the extraction conditions in step S1 are: 22 to 28 ℃, 170 to 230rpm, 1.5 to 2.5 hours.
According to the method of the present invention, the extraction conditions in step S1 are 25 ℃, 200rpm, and 2 h.
According to the method of the present invention, the conditions of the freeze-drying in step S2 are: the temperature is-70 to-80 ℃, and the time is 68 to 80 hours.
Preferably, according to the method of the present invention, the conditions of the freeze-drying in step S2 are: the temperature is-80 ℃ and the time is 72 h.
According to the method of the invention, the temperature of the storage in step S2 is-18 to-22 ℃.
Preferably, according to the method of the present invention, the temperature of the storage in step S2 is-20 ℃.
According to the method of the present invention, the total carbon content in step S2 is determined by a TOC analyzer.
According to the method of the invention, step S313The C content determination specifically comprises the steps of putting the powder into a tin cup for testing, wrapping the tin cup, and putting the wrapped tin cup into an isotope mass spectrometer for determination.
The invention has the beneficial effects that:
the volume of the sample to be detected can be greatly reduced through freeze drying, compared with the volume of the solution, the volume of the powder obtained by freezing is only about 1/5, so that the content of carbon isotopes in the sample in unit volume can be effectively improved, the sample injection concentration is higher than the detection limit of an instrument, the sample with lower concentration can be detected, the method is convenient and quick, the sensitivity is high, and the minimum concentration of the sample detectable by using the method is 0.5 mg/L.
Detailed Description
The present invention will be further illustrated by the following examples, but is not limited thereto.
Example 1
A method for determining the carbon isotope content of soil microorganisms comprises the following steps:
s1: a soil sample of 0-15 cm is collected from a field block of an experimental base of Yangtze field village in an urban-increasing area of Guangzhou, Guangdong province, and after stone and plant roots are removed, the soil sample is air-dried and sieved by a 4mm sieve for later use. With and without fumigation, a single replicate was added to a 10g soil sample (dry weight basis). And (4) placing the fumigated sample in a chloroform environment, and carrying out closed culture for 24 h. Followed by 50mL0.5mol/L of K2SO4The solution extracts soluble organic carbon in soil, wherein the extraction conditions are as follows: the temperature is 25 ℃, the rotation speed is 200rpm, and the time is 2 h. Directly using 50mL0.5mol/L K without fumigating to process samples2SO4And (4) extracting the solution. Each treatment was in 3 replicates.
S2: after the extraction, 10mL of the filtrate was used to determine the total carbon content using a TOC analyzer.
S3: and (3) placing 50mL of the mixture into a plastic centrifugal tube, sealing the tube opening by using a sealing strip, and pricking 3-4 small holes on the sealing strip of the tube opening by using a fine needle. The dehydration was carried out immediately using a freeze dryer (-80 ℃, 72 h). Immediately after dehydration, the resulting powder was placed in a sealed sample tube and stored frozen at-20 ℃ for later testing. During testing, 0.8-1.2 mg of powder is taken out, a tin cup for testing is added, the tin cup is wrapped into a solid sphere, and the solid sphere is placed into an isotope mass spectrometer for measuring in a sample13And (4) C content.
S4: calculating the microorganisms of the soil sample by the following formula13The content of C is as follows,
Figure BDA0002737177900000031
wherein the content of the first and second substances,13C-MBC is the carbon isotope content, delta, of soil microorganisms13Cf and delta13Cnf is freeze-dried solid sample treated with fumigation and without fumigation respectively13C abundance, Cf and Cnf are the total carbon content of the filtrates with and without fumigation treatment, respectively.
The same soil sample was collected and steps S1-S4 were repeated 3 times to test the stability of the test method by analyzing the data of different batches.
Specific results are shown in table 1.
TABLE 1 soil microorganisms in Yangtze village test base13C content detection result
Figure BDA0002737177900000032
Example 2
A method for determining the carbon isotope content of soil microorganisms comprises the following steps:
s1: a soil sample of 0-15 cm is collected from a field block of a six-grid village experiment base in east town of Reyowa, Zhanjiang, Guangdong province, stone and plant roots are removed, and the soil sample is air-dried and sieved by a 4mm sieve for later use. With and without fumigation, a single replicate was added to a 10g soil sample (dry weight basis). And (4) placing the fumigated sample in a chloroform environment, and carrying out closed culture for 24 h. Then 50mL of 0.5mol/L K is used2SO4The solution extracts soluble organic carbon in soil, wherein the extraction conditions are as follows: the temperature is 25 ℃, the rotation speed is 200rpm, and the time is 2 h. Directly using 50mL of 0.5mol/L K without fumigating to process a sample2SO4And (4) extracting the solution. Each treatment was in 3 replicates.
S2: after the extraction, 10mL of the filtrate was used to determine the total carbon content using a TOC analyzer.
S3: and (3) placing 50mL of the mixture into a plastic centrifugal tube, sealing the tube opening by using a sealing strip, and pricking 3-4 small holes on the sealing strip of the tube opening by using a fine needle. The dehydration was carried out immediately using a freeze dryer (-80 ℃, 72 h). Immediately after dehydration, the resulting powder was placed in a sealed sample tube and stored frozen at-20 ℃ for later testing. During testing, 0.8-1.2 mg of powder is taken out, a tin cup for testing is added, the tin cup is wrapped into a solid sphere, and the solid sphere is placed into an isotope mass spectrometer for measuring in a sample13And (4) C content.
S4: calculating the microorganisms of the soil sample by the following formula13The content of C is as follows,
Figure BDA0002737177900000041
wherein the content of the first and second substances,13C-MBC is the carbon isotope content, delta, of soil microorganisms13Cf and delta13Cnf is freeze-dried solid sample treated with fumigation and without fumigation respectively13C abundance, Cf and Cnf are total carbon in the filtrate treated with and without fumigation respectivelyAnd (4) content.
The same soil sample was collected and steps S1-S4 were repeated 3 times to test the stability of the test method by analyzing the data of different batches.
The specific results are shown in Table 2.
TABLE 2 soil microorganisms in six-Gecun Experimental base13C content detection result
Figure BDA0002737177900000042
Comparative example 1
A method for determining the carbon isotope content of soil microorganisms comprises the following steps:
1: a soil sample of 0-15 cm is collected from a field block of an experimental base of Yangtze field village in an urban-increasing area of Guangzhou, Guangdong province, and after stone and plant roots are removed, the soil sample is air-dried and sieved by a 4mm sieve for later use. With and without fumigation, a single replicate was added to a 10g soil sample (dry weight basis). And (4) placing the fumigated sample in a chloroform environment, and carrying out closed culture for 24 h. Then 50mL of 0.5mol/L K is used2SO4The solution extracts soluble organic carbon in soil, wherein the extraction conditions are as follows: the temperature is 25 ℃, the rotation speed is 200rpm, and the time is 2 h. Directly using 50mL of 0.5mol/L K without fumigating to process a sample2SO4And (4) extracting the solution. Each treatment was in 3 replicates.
2: after the extraction, 10mL of the filtrate was used to determine the total carbon content using a TOC analyzer.
3: and taking 50mL of the residual filtrate, and directly carrying out liquid carbon isotope determination by using a TOC-PDZ isotope mass spectrometer.
Collecting the same soil sample, repeating the steps 1-3 for 3 times, and testing the stability of the detection method by analyzing data of different batches.
The specific results are shown in Table 3.
TABLE 3 soil microorganisms in Yangtze village test base13C content detection result
Figure BDA0002737177900000051
Comparative example 2
1: a soil sample of 0-15 cm is collected from a field block of a six-grid village experiment base in east town of Reyowa, Zhanjiang, Guangdong province, stone and plant roots are removed, and the soil sample is air-dried and sieved by a 4mm sieve for later use. With and without fumigation, a single replicate was added to a 10g soil sample (dry weight basis). And (4) placing the fumigated sample in a chloroform environment, and carrying out closed culture for 24 h. Then extracting the soluble organic carbon in the soil by 50mL of 0.5mol/L K2SO4 solution, wherein the extraction conditions are as follows: the temperature is 25 ℃, the rotation speed is 200rpm, and the time is 2 h. Samples were directly extracted with 50mL of 0.5mol/L K2SO4 solution without fumigation. Each treatment was in 3 replicates.
2: after the extraction, 10mL of the filtrate was used to determine the total carbon content using a TOC analyzer.
3: and taking 50mL of the residual filtrate, and directly carrying out liquid carbon isotope determination by using a TOC-PDZ isotope mass spectrometer.
Collecting the same soil sample, repeating the steps 1-3 for 3 times, and testing the stability of the detection method by analyzing data of different batches.
The specific results are shown in Table 4.
TABLE 4 soil microorganisms in six-Gecun Experimental base13C content detection result
Figure BDA0002737177900000052
Example 3 sensitivity detection
1: using commercial glucose as carbon source (13The content of C is 1.0977 +/-0.0011 thousandths), a plurality of treatments of 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20 and 30mg/L (calculated by carbon, the content of carbon in the solution) are respectively set, and 3 parallel samples are set in each treatment. Dissolving glucose in 50mL of 0.5mol/L K according to the proportion2SO4In the solution, the solution after extraction was simulated, and mg/L (in terms of carbon) was used as a background value control.
2: placing the obtained solution in a plastic centrifugal tube, and sealing with a sealing stripAnd sealing the pipe orifice, and pricking 3-4 small holes on the pipe orifice sealing strip by using a fine needle. The dehydration was carried out immediately using a freeze dryer (-80 ℃, 72 h). Immediately after dehydration, the resulting powder was placed in a sealed sample tube and stored frozen at-20 ℃ for later testing. During testing, 0.8-1.2 mg of powder is taken out, a tin cup for testing is added, the tin cup is wrapped into a solid sphere, and the solid sphere is placed into an isotope mass spectrometer for measuring in a sample13And (4) C content.
3: the specific results are shown in Table 5.
TABLE 5 carbon content of different solutions13Detected value of C content
Figure BDA0002737177900000061
The results show that: when the carbon content of the solution is more than 0.5mg/L, more reliable data can be obtained. When the carbon content of the solution is lower than 0.5mg/L, effective data is difficult to obtain, which is closely related to an inspection instrument, so that the detection limit is set to 0.5 mg/L.
Example 4 accuracy analysis
(1) The results of the measurements of example 1 and comparative example 1 were analyzed for differences, in accordance with the ANOVA statistical analysis method, and are shown in Table 6, where P > 0.05. The two results are not statistically different, and the results can be judged to be consistent. The cost of the check analysis is only 23% of that of the direct liquid determination method.
Table 6 analysis of the difference between the measurement results of example 1 and comparative example 1
Figure BDA0002737177900000062
Figure BDA0002737177900000071
(2) The results of the measurements of example 2 and comparative example 2 were analyzed for differences, referring to the statistical analysis method of ANOVA, and the results are shown in Table 7, where P > 0.05. The two results are not statistically different, and the results can be judged to be consistent. The cost of the check analysis is only 23% of that of the direct liquid determination method.
Table 7 differential analysis of measurement results of example 2 and comparative example 2
Figure BDA0002737177900000072
The above embodiments are merely preferred examples to illustrate the present invention, and it should be apparent to those skilled in the art that any obvious variations and modifications can be made without departing from the spirit of the present invention.

Claims (10)

1. A method for determining the carbon isotope content of soil microorganisms comprises the following steps:
s1: dividing the soil samples into two groups, wherein the first group of samples are fumigated by chloroform and then extracted by potassium sulfate solution to obtain filtrate A, and the second group of samples are extracted by potassium sulfate solution to obtain filtrate B;
s2: taking a small amount of the total carbon content of the filtrate A and the filtrate B, respectively sealing the rest part, freeze-drying to obtain powder A and powder B, and freeze-preserving;
s3: respectively carrying out the steps of13C content is measured, and then the carbon isotope content of the soil microorganisms is calculated.
2. The method of claim 1, wherein the specific method for calculating the carbon isotope content of the soil microorganisms in step S3 is as follows: by passing
Figure FDA0002737177890000011
Calculating microorganisms of soil samples13C content;
wherein the content of the first and second substances,13C-MBC is the carbon isotope content, delta, of soil microorganisms13Cf and delta13Cnf for powder A and powder B respectively13C FengDegree, Cf and Cnf are the total carbon content of filtrate a and filtrate B, respectively.
3. The method of claim 1, wherein the soil sample is obtained by sieving a 4mm sieve in step S1.
4. The method as claimed in claim 1, wherein the chloroform fumigation treatment in step S1 is carried out by hermetically culturing the sample in chloroform environment for 20-28 h.
5. The method of claim 1, wherein the potassium sulfate concentration in step S1 is 0.4-0.6 mol/L.
6. The method of claim 1, wherein the soil-to-water ratio of the first set of samples to the potassium sulfate solution in step S1 is 1 (4-6).
7. The method of claim 1, wherein the soil-to-water ratio of the second set of samples to the potassium sulfate solution in step S1 is 1 (4-6).
8. The method of claim 1, wherein the extraction conditions in step S1 are: 22 to 28 ℃, 170 to 230rpm, 1.5 to 2.5 hours.
9. The method according to claim 1, wherein the freeze-drying conditions in step S2 are: the temperature is-70 to-80 ℃, and the time is 68 to 80 hours.
10. The method according to claim 1, wherein the temperature of the storing in step S2 is-18 to-22 ℃.
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