Method suitable for measuring contents of carbon, nitrogen and phosphorus of microorganisms in dry and wet soil

Abstract

The invention relates to the technical field of soil detection, and discloses a method suitable for measuring the content of carbon, nitrogen and phosphorus in dry and wet soil, an improved chloroform-extractant two-step extraction method is adopted, so that errors generated when a high-water-content sample is measured by a traditional chloroform fumigation-extraction method are effectively avoided, the problem of low measured value is solved, and the method is suitable for measuring the content of carbon, nitrogen and phosphorus in dry and wet soil. And the accuracy and the reliability of measurement are obviously improved. Compared with the prior art, the two-step extraction method provided by the invention is simpler and more convenient to operate, does not need complex fumigation equipment, only needs simple extraction steps, reduces the requirements on the equipment, reduces the operation steps and improves the working efficiency. The reaction conditions are normal temperature and normal pressure, the requirement on equipment is low, the dosage of chemicals is small, the applicable sample size range is wider, the operation is simple, and the measurement cost is low. The method is not only suitable for dry soil samples, but also suitable for wet and even high-moisture-content soil samples, and the application range of the measurement technology is greatly widened.

Description

Technical Field

[0001] The invention belongs to the technical field of soil detection, and in particular relates to a method for determining the contents of microbial carbon, nitrogen and phosphorus in dry and wet soils. Background Art

[0002] The content of soil microbial carbon, nitrogen and phosphorus is of great significance for soil fertility management and agricultural production. It is one of the important indicators for evaluating the health status of soil ecosystems and soil quality. It can provide a scientific basis for soil quality improvement and provide technical support for soil health monitoring and soil protection.

[0003] The mainstream detection method for soil microbial carbon, nitrogen and phosphorus content is the chloroform fumigation-extractant extraction method. This method places ethanol-free chloroform in dark conditions, uses a vacuum environment to generate chloroform vapor, and completely exposes the surface of sieved (200 mesh) air-dried soil particles to the chloroform environment, destroying the microbial cell membrane structure and releasing intracellular substances, which are then extracted into a solution using an extractant for subsequent measurement. This method has high accuracy for measuring soils with a moisture content of less than 30%, such as dry land, grassland, and vegetable fields.

[0004] The soil moisture content varies significantly depending on the environmental medium. The soil in paddy fields, river and lake sediments, wetlands and other environments has been submerged for a long time, has a high moisture content, and the soil particles are cohesive to each other, making it difficult to screen, which does not meet the sample requirements of the current method. If the current method is still used for measurement, although the vacuum environment can generate chloroform vapor, it is also easy to freeze the water, forming a solid film covering the surface of the sample particles, which prevents the chloroform vapor from contacting the sample surface. In addition, the chloroform vapor diffuses from top to bottom and from outside to inside into the sample. When it contacts the surface of the high-water content sample, it is easy to condense into a liquid state, and diffuse outward and upward to form vapor again, which is difficult to contact the internal sample, resulting in incomplete and uneven fumigation, affecting the measurement results. If the sample is air-dried, it takes a long time and causes microbial proliferation. If it is blown or dried, centrifuged, etc., the splashing droplets are easy to carry away the biofilm formed by microorganisms on the surface of the stripping filler, affecting the measurement results. On the other hand, the safe operating range of commonly used vacuum dryers is ≤0.06 MPa, while the current method requires a fumigation vacuum degree of 0.05-0.08 MPa, which places high demands on experimental equipment. In addition, the formation rate of chloroform vapor during the fumigation process (i.e., vapor concentration) varies with the vacuum degree, which may cause different degrees of cell membrane damage and affect the efficiency of fumigation and subsequent extraction. Summary of the invention

[0005] The purpose of the present invention is to overcome the above problems and provide a method for determining the content of microbial carbon, nitrogen and phosphorus in dry and wet soils. The method has low equipment requirements, simple operation, and is applicable to both dry and wet samples.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: The present invention provides a method for determining the content of microbial carbon, nitrogen and phosphorus in dry and wet soil, comprising the following steps: S1, chloroform extraction: after measuring the moisture content of the sample, weigh three portions of the sample to be tested, each of which is equivalent to 10 g of dry weight, and place them in glass centrifuge tubes respectively. Add chloroform directly to the surface of the first portion, tighten the cap of the glass centrifuge tube and place it in a dark environment for chloroform extraction; do not add chloroform to the second portion, tighten the cap of the glass centrifuge tube and place it in a dark environment; do not do any treatment to the third sample.

[0007] S2, extraction with extractant: After the chloroform extraction is completed, the extractant is injected into each of the three samples, and then a known content of carbon, nitrogen or phosphorus is injected into the third sample, and the samples are placed in a cyclotron for shaking and mixing to extract microbial carbon, nitrogen or phosphorus; The third sample is used to determine the extraction recovery rate, and the difference between the first and second samples is the microbial carbon, nitrogen or phosphorus content after recovery rate calibration.

[0008] A further improvement of the present invention is that the chloroform extraction is carried out in a light-proof environment at normal temperature and pressure, and the extraction time is 24 hours.

[0009] A further improvement of the present invention is that the minimum amount of chloroform is 50-80 μL / g dry sample or is added in excess.

[0010] A further improvement of the present invention is that the extractant for microbial carbon and nitrogen is 0.5M potassium persulfate, and the extractant for microbial phosphorus is 0.5M sodium bicarbonate, and the dosage of the extractants is 10ml / g dry sample.

[0011] A further improvement of the present invention is that 100 ml glass centrifuge tubes are used in both the chloroform extraction and the extractant extraction processes.

[0012] A further improvement of the present invention is that the chloroform extraction and the extractant extraction are both subjected to cyclotron oscillation at 150-250 r / min for 25-35 min.

[0013] A further improvement of the present invention is that the method for determining the microbial carbon content is as follows: a 0.5 M potassium persulfate solution is injected into the sample after chloroform extraction at a rate of 10 ml / g of dry sample and vortexed to extract carbon, and then the microbial carbon sample and the glass centrifuge tube are immersed in a boiling water bath, filtered, and the total organic carbon content is determined in a total organic carbon analyzer, and the difference in sample detection with and without chloroform added is the microbial carbon content after recovery calibration.

[0014] A further improvement of the present invention is that the microbial carbon sample is immersed in a boiling water bath for 90 minutes; and the measuring temperature of the total organic carbon measuring instrument is 800-1200°C.

[0015] A further improvement of the present invention is that the method for determining the microbial nitrogen content is as follows: a 0.5M potassium persulfate solution is injected into the sample after chloroform extraction at a rate of 10 ml / g of dry sample and vortexed to extract nitrogen; after filtration, the total nitrogen content is detected by alkaline potassium persulfate digestion ultraviolet spectrophotometry; the difference between the sample detections with and without chloroform added is calibrated by recovery rate to obtain the microbial nitrogen content.

[0016] A further improvement of the present invention is that the method for determining the microbial phosphorus content is as follows: a 0.5M sodium bicarbonate solution is injected into the sample after chloroform extraction at a rate of 10 ml / g of dry sample and vortexed to extract phosphorus; after the extraction is completed, hydrochloric acid is added thereto, and after filtering, the total phosphorus content is detected by ammonium molybdate spectrophotometry; the difference in the sample detection between the addition of chloroform and the addition of no chloroform is calibrated with the recovery rate to obtain the microbial phosphorus content.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for determining the content of microbial carbon, nitrogen and phosphorus in dry and wet soils. The improved chloroform-extractant two-step extraction method effectively avoids the error caused by the traditional chloroform fumigation-extraction method when measuring high-water content samples, overcomes the problem of low measured values, and significantly improves the accuracy and reliability of the measurement. Compared with the existing method, the two-step extraction method of the present invention is simpler to operate, does not require complex fumigation equipment, and only requires simple extraction steps, which reduces the requirements for equipment, reduces the operating steps, and improves work efficiency. The present method is not only applicable to dry soil samples, but also to moist and even high-water content soil samples, greatly broadening the application scope of the measurement technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings described herein are only for explanation purposes and are not intended to limit the scope of the present invention in any way. In addition, the shapes and proportional dimensions of the various components in the drawings are only schematic, used to help understand the present invention, and are not intended to specifically limit the shapes and proportional dimensions of the various components of the present invention.

[0019] Figure 1 The test method for chloroform fumigation and leaching in the current method; Figure 2 The improved chloroform extraction and extract extraction test methods of the present invention; Figure 3 is a graph showing the relationship between the amount of chloroform used in the present invention and the microbial carbon content in different soils; Figure 4 is a graph showing the relationship between the amount of chloroform used in the present invention and the microbial nitrogen content in different soils; Figure 5 is a graph showing the relationship between the amount of chloroform used in the present invention and the phosphorus content of microorganisms in different soils; Figure 6 It is a relationship diagram between the amount of extractant used and the measured value of microbial carbon content in the present invention; Figure 7 It is a relationship diagram between the amount of extractant used in the present invention and the measured value of microbial nitrogen content; Figure 8 It is a relationship diagram between the amount of the extractant used in the present invention and the measured value of the phosphorus content of the microorganism; Fig. 9 The effect of water bathing on the total organic carbon content of different soils in the present invention; Fig.10 It is a relationship diagram between the water bath time and the residual amount of chloroform in the present invention; Fig.11 To compare the test results of microbial carbon content in dry samples between the improved method and the current method; Fig.12 To compare the test results of microbial nitrogen content in dry samples between the improved method and the current method; Fig.13 To compare the test results of microbial phosphorus content in dry samples between the improved method and the current method; Fig.14 The results of the microbial carbon content test of dry and wet samples in the improved method are compared; Fig.15 The comparison of the test results of microbial nitrogen content between dry and wet samples in the improved method is presented; Fig.16 This is a comparison of the test results of microbial phosphorus content in dry samples and wet samples using the improved method. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0021] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0022] In this article, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0023] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0024] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0025] The present invention provides a method for determining the content of microbial carbon, nitrogen and phosphorus in dry and wet soil, comprising the following steps: Step S1, weigh three samples to be tested, each equivalent to 5-10g of dry weight, and place them in 100ml glass centrifuge tubes respectively, add chloroform to the surface of the first sample at a dosage of 50-80μL / g, and do not add chloroform to the second sample; then tighten the caps of the glass centrifuge tubes of the two samples and place them in a light-proof environment at room temperature and pressure for 24 hours; after tightening the cap of the third sample, do not do any treatment.

[0026] It should be noted that a chloroform addition amount of 50-80 μL / g dry weight can completely extract microbial carbon, nitrogen and phosphorus. Excessive chloroform can also be added to completely immerse the sample in chloroform. This practice does not affect the extraction efficiency.

[0027] Step S2, injecting an extractant into the three samples at a rate of 10 ml / g of dry sample, adding a known content of carbon, nitrogen or phosphorus into the third sample, placing the three samples in a cyclotron oscillator at 150-250 r / min for 25-35 min to extract carbon, nitrogen or phosphorus from the microorganisms; the third sample is used to determine the recovery rate of the extractant extraction, and the detection difference between the first two samples is the microbial carbon, nitrogen or phosphorus content after recovery rate calibration.

[0028] Among them, the method for determining the microbial carbon content is as follows: after S1 is completed, bovine serum albumin equivalent to 1 mg / L of total organic carbon is added to the third sample; 0.5M potassium persulfate solution is injected into the three samples at a rate of 10 ml / g dry sample and placed in a cyclotron oscillator at 150~250 r / min for 25~35 minutes to extract carbon, and then the microbial carbon sample and the glass centrifuge tube are immersed in a boiling water bath for 90 minutes, and after filtration, the total organic carbon content is determined at a temperature of 800~1200°C in a total organic carbon analyzer; the measured value of the third sample is used to calculate the recovery rate, and the detection difference of the other two samples is the microbial carbon content after recovery rate calibration.

[0029] The method for determining the microbial nitrogen content is as follows: after S1 is completed, sodium nitrate equivalent to 0.3 mg N / L is added to the third sample; 0.5 M potassium persulfate solution is injected into the three samples at a rate of 10 ml / g dry sample and placed in a cyclotron oscillator at 150-250 r / min for 25-35 min to extract nitrogen; after filtration, the total nitrogen content is detected by the alkaline potassium persulfate digestion ultraviolet spectrophotometry method according to GB11894-89; the detection value of the third sample is used to determine the recovery rate, and the detection difference of the other two samples is the microbial nitrogen content after correction by the recovery rate.

[0030] The method for determining the microbial phosphorus content is as follows: after S1 is completed, potassium dihydrogen phosphate equivalent to 0.1 mgN / L is added to the third sample; 0.5 M sodium bicarbonate solution is injected into the three samples at a rate of 10 ml / g dry sample and placed in a cyclotron oscillator at 150-250 r / min for 25-35 min to extract phosphorus. After the extraction is completed, sufficient hydrochloric acid is added thereto to convert sodium bicarbonate into a product that does not affect subsequent measurements, and the samples are oscillated at 150-250 r / min for 10-20 min to expel carbon dioxide. After filtration, the total phosphorus content is detected according to the ammonium molybdate spectrophotometry method according to GB11893-89; the measured value of the third sample is used to calculate the recovery rate, and the detection difference of the other two samples is the microbial phosphorus content after correction by the recovery rate.

[0031] In this improved method, chloroform is heavier than water and has strong volatility. After chloroform (AR) is dripped in, chloroform quickly evaporates while settling down along the sample pores, forming chloroform vapor in the space above the sample. Chloroform can corrode plastic, causing the organic matter in the commonly used plastic centrifuge tube to be released into the soil sample, causing measurement errors. Therefore, glass centrifuge tubes must be used. The chloroform that settles inside the sample forms vapor and diffuses to the surrounding pores. The diffused chloroform vapor contacts the surface of the sample particles and condenses into liquid chloroform again. After repeated cycles, it is evenly distributed inside the sample. Chloroform is heavier than water. During its diffusion process, it continuously peels off the water film on the surface of the sample particles and moves it upward. The chloroform vapor inside the glass centrifuge tube finally completely covers the surface of the sample particles, achieving a uniform and thorough fumigation effect, overcoming the defect of the current method that the liquid film on the sample surface freezes to form a solid coating that affects the measurement results. After chloroform extraction, the microbial cell membrane is damaged and the intracellular substances are released, resulting in a difference with the unextracted sample. This difference is the microbial carbon, nitrogen and phosphorus content in the sample after recovery calibration.

[0032] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0033] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples are conventional commercial products unless otherwise specified, and their specifications are conventional specifications in the art. In the specification of the present invention and the following examples, unless otherwise specified, "%" means weight percentage, "part" means weight part, and ratio means weight ratio.

[0034] Example 1 This embodiment provides a method for determining the microbial carbon content in humus soil, comprising the following steps: Step 1, setting up five groups of completely identical parallel samples, taking the first group of samples as an example: weighing 200g of humus soil sample, dividing it into four parts, one of which is dried and measured to have a moisture content of 62%; taking 10g of humus soil equivalent to dry weight from the remaining three samples and placing them in 100ml glass centrifuge tubes respectively, dripping chloroform into the first glass centrifuge tube at a dosage of 500μL / g dry weight, that is, dripping 5ml of chloroform, covering it and shaking it vigorously; not dripping chloroform into the second glass centrifuge tube, directly covering it and sealing it; then placing the above two glass centrifuge tubes in dark conditions at room temperature and pressure for 24h; and not doing any treatment after the third glass centrifuge tube is covered.

[0035] Step 2, after the chloroform extraction is completed, take out three glass centrifuge tubes, open the lids and add 0.5M potassium persulfate solution to each of the glass centrifuge tubes at a rate of 10 ml / g dry sample, i.e., add 100 ml; Step 3, directly placing three glass centrifuge tubes in a cyclotron, adding bovine serum albumin equivalent to 1 mg / L total organic carbon to the third glass centrifuge tube, and placing it in a cyclotron after fully dissolving; and oscillating all three glass centrifuge tubes at 200r for 30min; Step 4, place the three glass centrifuge tubes in a boiling water bath for 90 min to drive out the residual chloroform; Step 5, filter the sample, collect the filtrate, use distilled water to repeatedly rinse the residual solid components 3-5 times to obtain an eluent, fully mix the liquid phase components and the eluent, and set the volume to 250 ml for subsequent measurement. The total organic carbon content is detected at 900°C in the total organic carbon meter; the sample detection value in the third glass centrifuge tube is used to calculate the recovery rate. In this embodiment, the recovery rate is 97.0% and the standard deviation is 3.32%. The difference between the measurement results of the first and second glass centrifuge tubes divided by the recovery rate is the microbial carbon content, that is, 610.5μg C / g dry weight, and the standard deviation is 3.16%. It should be noted that in this step, the dilution factor given by this method is 2.5 times. In actual measurement, the dilution factor, that is, the set volume capacity, can be flexibly adjusted according to the actual sample microbial carbon, nitrogen, and phosphorus content and sample detection requirements.

[0036] Example 2 This embodiment provides a method for determining the microbial nitrogen content in humus soil, comprising the following steps: Step 1, setting a total of five groups of completely identical parallel samples, taking the first group of samples as an example: weighing 200g of humus soil sample, dividing it into four parts, one of which is dried and measured to have a moisture content of 62%; taking 10g of humus soil equivalent to dry weight from the remaining three samples and placing them in 100ml glass centrifuge tubes respectively, dripping chloroform in the first glass centrifuge tube at a dosage of 500μL / g dry weight, that is, dripping 5ml of chloroform, covering and vigorously shaking to mix; not dripping chloroform in the second glass centrifuge tube, directly covering and sealing; then placing the above two glass centrifuge tubes in dark conditions at room temperature and pressure for 24h; the third glass centrifuge tube is covered without any treatment; Step 2, after the chloroform extraction is completed, take out three glass centrifuge tubes, open the lids and add 0.5M potassium persulfate solution to each of the glass centrifuge tubes at a rate of 10 ml / g dry sample, i.e., add 100 ml; Step 3, directly placing three glass centrifuge tubes in a cyclotron shaker, adding 0.3 mg N / L sodium nitrate (AR) to the third glass centrifuge tube, fully dissolving and placing in a cyclotron shaker; shaking all three glass centrifuge tubes at 200 r for 30 min; Step 4, filter the sample, collect the filtrate, and detect the total nitrogen content according to GB11894-89. Use distilled water to repeatedly rinse the residual solid components 3-5 times to obtain an eluent, fully mix the liquid phase components and the eluent, and set the volume to 250ml for subsequent measurement. The measurement result of the third glass centrifuge tube is used to calculate the recovery rate. In this embodiment, the recovery rate is 99% and the standard deviation is 3.16%. The difference in the measurement results of the first and second glass centrifuge tubes is calibrated with the recovery rate to obtain the microbial nitrogen content, i.e. 60.9μg N / g, and the standard deviation is 4.56%. It should be noted that in this step, the dilution factor given by this method is 2.5 times. In actual measurement, the dilution factor, i.e., the set volume capacity, can be flexibly adjusted according to the actual sample microbial carbon, nitrogen, phosphorus content and sample detection requirements.

[0037] Example 3 This embodiment provides a method for determining the phosphorus content of microorganisms in humus soil, comprising the following steps: Step 1, setting a total of five groups of completely identical parallel samples, taking the first group of samples as an example: weighing 200g of humus soil sample, dividing it into four parts, one of which is dried and measured to have a moisture content of 62%; taking 10g of humus soil equivalent to dry weight from the remaining three samples and placing them in 100ml glass centrifuge tubes respectively, dripping chloroform in the first glass centrifuge tube at a dosage of 500μL / g dry weight, that is, dripping 5ml of chloroform, covering and vigorously shaking to mix; not dripping chloroform in the second glass centrifuge tube, directly covering and sealing; then placing the above two glass centrifuge tubes in dark conditions at room temperature and pressure for 24h; the third glass centrifuge tube is not treated in any way; Step 2, after the chloroform extraction is completed, take out three glass centrifuge tubes, open the lids and add 0.5M sodium bicarbonate solution to the glass centrifuge tubes at 10ml / g dry sample, i.e., add 100ml, and then add half the dose of 1M hydrochloric acid, i.e., add 50ml, and oscillate at 200r in a cyclotron for 15min to expel carbon dioxide; Step 3, directly place three glass centrifuge tubes in a cyclotron, add potassium dihydrogen phosphate (AR) equivalent to 0.1 mgP / L to the third glass tube, fully dissolve it and place it in a cyclotron; shake all three glass centrifuge tubes at 200r for 30min; Step 4, filter the sample, collect the filtrate, and detect the total phosphorus content according to the method described in GB11893-89. Use distilled water to repeatedly rinse the residual solid component 3-5 times to obtain an eluent, fully mix the liquid phase component and the eluent, and set the volume to 250ml for subsequent measurement. The measurement result of the third glass centrifuge tube is used to calculate the recovery rate. In this embodiment, the recovery rate is 97% and the standard deviation is 2.89%. The difference between the measurement results of the first and second glass centrifuge tubes divided by the recovery rate is the microbial phosphorus content, i.e. 4.63μg P / g, and the standard deviation is 4.69%. It should be noted that in this step, the dilution factor given by this method is 2.5 times. In actual measurement, the dilution factor, i.e., the set volume capacity, can be flexibly adjusted according to the actual sample microbial carbon, nitrogen, phosphorus content and sample detection requirements.

[0038] Example 4 This embodiment provides a method for determining the microbial carbon content in a vegetable field, comprising the following steps: A total of five groups of identical parallel samples were set up. Taking the first group of samples as an example: 200g of vegetable soil sample was weighed and divided into four equal parts, one of which was dried and its moisture content was determined to be 43%; the treatment and other steps of the remaining three samples were the same as in Example 1. In this example, the recovery rate was 98.3% and the standard deviation was 4.72%. The difference between the measurement results of the first and second glass centrifuge tubes divided by the recovery rate is the microbial carbon content, that is, 467.2μg C / g dry weight, and the standard deviation is 3.80%.

[0039] Example 5 This embodiment provides a method for determining the microbial nitrogen content in a vegetable field, comprising the following steps: A total of five groups of identical parallel samples were set up. Taking the first group of samples as an example: 200g of vegetable soil sample was weighed and divided into four equal parts, one of which was dried and its moisture content was determined to be 43%; the treatment and other steps of the remaining three samples were the same as in Example 2. In this example, the recovery rate was 97.6% and the standard deviation was 3.21%. The difference between the measurement results of the first and second glass centrifuge tubes divided by the recovery rate is the microbial nitrogen content, that is, 44.3μg N / g dry weight, and the standard deviation is 4.23%.

[0040] Example 6 This embodiment provides a method for determining the phosphorus content of microorganisms in a vegetable field, comprising the following steps: A total of five groups of identical parallel samples were set up. Taking the first group of samples as an example: 200g of vegetable soil sample was weighed and divided into four equal parts, one of which was dried and its moisture content was determined to be 43%; the treatment and other steps of the remaining three samples were the same as in Example 3. In this example, the recovery rate was 99.4% and the standard deviation was 1.06%. The difference between the measurement results of the first and second glass centrifuge tubes divided by the recovery rate is the microbial phosphorus content, that is, 4.96μg P / g dry weight, and the standard deviation is 3.66%.

[0041] Example 7 This embodiment provides a method for determining the microbial carbon content in a river bank, comprising the following steps: A total of five groups of identical parallel samples were set up. Taking the first group of samples as an example: 200g of riverbank soil samples were weighed and divided into four equal parts, one of which was dried and its moisture content was determined to be 62%; the treatment and other steps of the remaining three samples were the same as in Example 1. In this example, the recovery rate was 101.2% and the standard deviation was 4.34%. The difference between the measurement results of the first and second glass centrifuge tubes divided by the recovery rate is the microbial carbon content, that is, 157.4μg C / g dry weight, and the standard deviation is 4.34%.

[0042] Example 8 This embodiment provides a method for determining the microbial nitrogen content in a river bank, comprising the following steps: A total of five groups of identical parallel samples were set up. Taking the first group of samples as an example: 200g of riverbank soil samples were weighed and divided into four equal parts, one of which was dried and its moisture content was 62%; the treatment and other steps of the remaining three samples were the same as in Example 2. In this example, the recovery rate was 99.4% and the standard deviation was 2.06%. The difference between the measurement results of the first and second glass centrifuge tubes divided by the recovery rate is the microbial nitrogen content, that is, 14.8μg N / g dry weight, and the standard deviation is 4.71%.

[0043] Example 9 This embodiment provides a method for determining the phosphorus content of microorganisms in a river bank, comprising the following steps: A total of five groups of identical parallel samples were set up. Taking the first group of samples as an example: 200g of riverbank soil samples were weighed and divided into four equal parts, one of which was dried and its moisture content was 62%; the treatment and other steps of the remaining three samples were the same as in Example 3. In this example, the recovery rate was 102.5% and the standard deviation was 4.32%. The difference between the measurement results of the first and second glass centrifuge tubes divided by the recovery rate is the microbial phosphorus content, that is, 1.22μg P / g dry weight, and the standard deviation is 4.16%.

[0044] Example 10 This embodiment provides a method for determining the microbial carbon content in wasteland soil next to an infiltration pit, comprising the following steps: A total of five groups of identical parallel samples were set up. Taking the first group of samples as an example: 200g of wasteland soil sample next to the infiltration pit was weighed and divided into four equal parts, one of which was dried and its moisture content was 57%; the treatment and other steps of the remaining three samples were the same as in Example 1. In this example, the recovery rate was 96.7% and the standard deviation was 2.34%. The difference between the measurement results of the first and second glass centrifuge tubes divided by the recovery rate is the microbial carbon content, that is, 518.8μg C / g dry weight, and the standard deviation is 3.10%.

[0045] Embodiment 11 This embodiment provides a method for determining the microbial nitrogen content in wasteland soil next to an infiltration pit, comprising the following steps: A total of five groups of identical parallel samples were set up. Taking the first group of samples as an example: 200g of wasteland soil sample next to the infiltration pit was weighed and divided into four equal parts, one of which was dried and its moisture content was determined to be 57%; the treatment and other steps of the remaining three samples were the same as in Example 2. In this example, the recovery rate was 98.9% and the standard deviation was 3.32%. The difference between the measurement results of the first and second glass centrifuge tubes divided by the recovery rate is the microbial nitrogen content, that is, 50.4 μg N / g dry weight, and the standard deviation is 4.62%.

[0046] Example 12 This embodiment provides a method for determining the microbial phosphorus content in wasteland soil next to an infiltration pit, comprising the following steps: A total of five groups of identical parallel samples were set up. Taking the first group of samples as an example: 200g of wasteland soil sample next to the infiltration pit was weighed and divided into four equal parts, one of which was dried and its moisture content was 57%; the treatment and other steps of the remaining three samples were the same as in Example 3. In this example, the recovery rate was 96.7% and the standard deviation was 3.84%. The difference between the measurement results of the first and second glass centrifuge tubes divided by the recovery rate is the microbial phosphorus content, that is, 4.23μg P / g dry weight, and the standard deviation is 4.14%.

[0047] Embodiment 13 This embodiment provides a method for determining the microbial carbon content in flower bed soil, comprising the following steps: A total of five groups of identical parallel samples were set up. Taking the first group of samples as an example: 200g of flower bed soil sample was weighed and divided into four equal parts, one of which was dried and its moisture content was determined to be 29%; the treatment and other steps of the remaining three samples were the same as in Example 1. In this example, the recovery rate was 99.1% and the standard deviation was 4.28%. The difference between the measurement results of the first and second glass centrifuge tubes divided by the recovery rate is the microbial carbon content, that is, 303.3μg C / g dry weight, and the standard deviation is 4.21%.

[0048] Embodiment 14 This embodiment provides a method for determining the microbial nitrogen content in flower bed soil, comprising the following steps: A total of five groups of identical parallel samples were set up. Taking the first group of samples as an example: 200g of flower bed soil sample was weighed and divided into four equal parts, one of which was dried and its moisture content was determined to be 29%; the treatment and other steps of the remaining three samples were the same as in Example 2. In this example, the recovery rate was 97.4% and the standard deviation was 2.63%. The difference between the measurement results of the first and second glass centrifuge tubes divided by the recovery rate is the microbial nitrogen content, that is, 34.5μg N / g dry weight, and the standard deviation is 3.08%.

[0049] Embodiment 15 This embodiment provides a method for determining the microbial phosphorus content in flower bed soil, comprising the following steps: A total of five groups of identical parallel samples were set up. Taking the first group of samples as an example: 200g of flower bed soil sample was weighed and divided into four equal parts, one of which was dried and its moisture content was determined to be 29%; the treatment and other steps of the remaining three samples were the same as in Example 3. In this example, the recovery rate was 101.5% and the standard deviation was 4.62%. The difference between the measurement results of the first and second glass centrifuge tubes divided by the recovery rate is the microbial phosphorus content, that is, 2.46μg P / g dry weight, and the standard deviation is 4.19%.

[0050] like Figures 3 to 5 As shown in the figure, under different soil sample environments, when the chloroform dosage was 10-300 μL / g dry sample, the release of microbial carbon gradually increased with the increase of chloroform dosage. When the chloroform dosage was 50-80 μL / g dry sample, the microbial carbon release was the highest and remained unchanged at higher dosages. The chloroform dosage of 50-80 μL / g dry sample can completely release the microbial carbon.

[0051] like Figures 6 to 8 As shown in the figure, in the flower bed soil environment, when the amount of extractant is 10ml / g dry sample, the microbial carbon, nitrogen and phosphorus content can reach the maximum extraction efficiency, which is only half of the current method, indicating that the improved method can maintain the maximum extraction efficiency while reducing the amount of extractant, which helps to reduce the extraction cost. It should be pointed out that under this extraction system, in 8 parallel samples, the recovery rates of carbon, nitrogen and phosphorus are between 96%-103%, 97%-104% and 96%-101%, respectively, which shows that the improved method has a high recovery rate and can accurately extract the target substance. In addition, the relative standard deviations are 3.32%, 5.60% and 5.37%, respectively, indicating that the improved method has a lower random error in the extraction process and the results are more stable and reliable. Moreover, the entire extraction process is carried out at room temperature and pressure, using a cyclotron oscillator to oscillate at 200r for 30min. Compared with the current method, a good extraction effect can still be obtained by reducing the oscillation speed by 100r.

[0052] Residual chloroform seriously affects the results of microbial carbon determination, so this improved method uses a boiling water bath to expel residual chloroform. Fig. 9 and Fig.10 As shown, it has been verified that whether or not to use a water bath for 90 minutes has no significant effect on the detection results of microbial carbon content in different samples; in this verification step, when 250ml of 0.5M potassium persulfate solution contains 40ml of chloroform, the chloroform can be completely expelled by a boiling water bath for 90 minutes. At this time, the amount of chloroform is equivalent to 4ml / g dry sample, which is far beyond the recommended amount. Therefore, the 90-minute water bath time can ensure that the chloroform is completely expelled when the amount of chloroform used is high.

[0053] like Figures 11 to 13 As shown, the improved method and the classical method were used to determine the microbial carbon, nitrogen and phosphorus contents of different soil samples in the above examples. Fig.11As shown in the figure, the results of the determination of microbial carbon content in different soil samples using the improved method are: flower bed 309.9, vegetable field 456.4, river bank 164.1, wasteland next to seepage pit 518.9, humus soil 620.5; the results of the determination of microbial carbon content in different soil samples using the classical method are: flower bed 305.3, vegetable field 452.2, river bank 167.3, wasteland next to seepage pit 517.6, humus soil 623.8. Fig.12 As shown in the figure, the results of the determination of microbial nitrogen content in different soil samples using the improved method are: flower bed 36.53, vegetable field 42.8, river bank 16.7, wasteland next to seepage pit 46.9, humus soil 62.6; the results of the determination of microbial nitrogen content in different soil samples using the classical method are: flower bed 35.37, vegetable field 43.3, river bank 15.4, wasteland next to seepage pit 47.5, humus soil 61.5. Fig.13 As shown in the figure, the results of the determination of microbial phosphorus content in different soil samples by the improved method were: flower bed 2.596, vegetable field 5.129, river bank 1.132, wasteland beside seepage pit 4.745, humus soil 4.831; the results of the determination of microbial phosphorus content in different soil samples by the classical method were: flower bed 2.446, vegetable field 4.982, river bank 1.492, wasteland beside seepage pit 4.249, humus soil 4.663. The results showed that there was no significant difference between the improved method and the classical method in dry sample measurement (p=0.233), indicating that the measurement results of the improved method were accurate and reliable.

[0054] like Figures 14 to 16 As shown, the improved method is used to determine the microbial carbon, nitrogen and phosphorus content in dry and wet samples of different soil samples in the above embodiment. Specifically, the soil sample dried to constant weight is divided into two parts, which are placed in glass centrifuge tubes respectively. Distilled water is added to one part until it just covers the surface of the soil sample to make a wet sample; the other soil sample is not treated in any way, and then the above method is used to determine the microbial carbon, nitrogen and phosphorus content in the dry and wet samples. Fig.14 As shown in the figure, the results of the determination of the microbial carbon content in dry samples of different soil samples using the improved method are: flower bed 223.8, vegetable field 415.7, river bank 133.8, wasteland next to seepage pit 536.6, humus soil 610.4; the results of the determination of the microbial carbon content in wet samples of different soil samples using the improved method are: flower bed 225.6, vegetable field 410.3, river bank 130.4, wasteland next to seepage pit 532.3, humus soil 617.6. Fig.15As shown in the figure, the results of the determination of microbial nitrogen content in dry samples of different soil samples using the improved method are: flower bed 33.6, vegetable field 42.3, river bank 14.8, wasteland next to seepage pit 48.3, humus soil 62.4; the results of the determination of microbial nitrogen content in wet samples of different soil samples using the improved method are: flower bed 31.8, vegetable field 44.5, river bank 13.6, wasteland next to seepage pit 49.6, humus soil 61.8. Fig.16 As shown in the figure, the results of the determination of microbial phosphorus content in dry samples of different soil samples using the improved method were: flower bed 2.587, vegetable field 5.016, river bank 1.788, wasteland beside seepage pit 4.317, humus 4.633; the results of the determination of microbial phosphorus content in wet samples of different soil samples using the improved method were: flower bed 2.297, vegetable field 4.941, river bank 1.507, wasteland beside seepage pit 4.158, humus 4.480. The results showed that there was no significant difference in the measurement results of dry and wet samples using this improved method (p=0.172).

[0055] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for determining the content of microbial carbon, nitrogen and phosphorus in dry and wet soils, characterized in that: The following steps are involved: S1, chloroform extraction: after measuring the moisture content of the sample, weigh three portions of the sample to be tested, each of which is equivalent to 10 g of dry weight, and place them in glass centrifuge tubes respectively. Add chloroform directly to the surface of the first portion, tighten the cap of the glass centrifuge tube and place it in a dark environment for chloroform extraction; do not add chloroform to the second portion, tighten the cap of the glass centrifuge tube and place it in a dark environment; do not do any treatment to the third portion; S2, extraction with extractant: After the chloroform extraction is completed, the extractant is injected into each of the three samples, and then a known content of carbon, nitrogen or phosphorus is injected into the third sample, and the samples are placed in a cyclotron for shaking and mixing to extract microbial carbon, nitrogen or phosphorus; The third sample is used to determine the extraction recovery rate, and the difference between the first and second samples is the microbial carbon, nitrogen or phosphorus content after recovery rate calibration.

2. A method for determining the content of microbial carbon, nitrogen and phosphorus in dry and wet soil according to claim 1, characterized in that: The chloroform extraction is carried out in a light-proof environment at normal temperature and pressure, and the extraction time is 24 hours.

3. A method for determining the content of microbial carbon, nitrogen and phosphorus in dry and wet soil according to claim 1, characterized in that: The amount of chloroform used is 50-80 μL / g dry sample or added in excess.

4. A method for determining the content of microbial carbon, nitrogen and phosphorus in dry and wet soil according to claim 1, characterized in that: The extractant for microbial carbon and nitrogen is 0.5M potassium persulfate, and the extractant for microbial phosphorus is 0.5M sodium bicarbonate, and the dosage of the extractants is 10ml / g dry sample.

5. A method for determining the content of microbial carbon, nitrogen and phosphorus in dry and wet soil according to claim 1, characterized in that: A 100 ml glass centrifuge tube was used in both the chloroform extraction and the extractant extraction.

6. A method for determining the content of microbial carbon, nitrogen and phosphorus in dry and wet soil according to claim 1, characterized in that: The chloroform extraction and the extractant extraction were both performed by vortex shaking at 150-250 r / min for 25-35 min.

7. A method for determining the content of microbial carbon, nitrogen and phosphorus in dry and wet soil according to claim 1, characterized in that: The method for determining the microbial carbon content is as follows: a 0.5 M potassium persulfate solution is injected into a sample that has been extracted with chloroform at a rate of 10 ml / g of dry sample and vortexed to extract carbon; the microbial carbon sample and a glass centrifuge tube are then immersed in a boiling water bath; after filtration, the total organic carbon content is determined in a total organic carbon analyzer; the difference in the sample detection value with and without chloroform added is calibrated with the recovery rate to obtain the microbial carbon content.

8. A method for determining the content of microbial carbon, nitrogen and phosphorus in dry and wet soil according to claim 7, characterized in that: The microbial carbon sample is immersed in a boiling water bath for 90 minutes; the measuring temperature of the total organic carbon measuring instrument is 800-1200°C.

9. A method for determining the content of microbial carbon, nitrogen and phosphorus in dry and wet soil according to claim 1, characterized in that: The method for determining the microbial nitrogen content is as follows: inject 0.5M potassium persulfate solution into the sample extracted with chloroform at a rate of 10 ml / g of dry sample and vortex to extract nitrogen; after filtering, detect the total nitrogen content by alkaline potassium persulfate digestion ultraviolet spectrophotometry; the difference in the sample detection value with or without chloroform added is the microbial nitrogen content after calibration with the recovery rate.

10. A method for determining the content of microbial carbon, nitrogen and phosphorus in dry and wet soil according to claim 1, characterized in that: The method for determining the phosphorus content of microorganisms is as follows: 0.5M sodium bicarbonate solution is injected into the sample after chloroform extraction at a rate of 10 ml / g dry sample and vortexed to extract phosphorus; after the extraction is completed, hydrochloric acid is added thereto, and after filtering, the total phosphorus content is detected by ammonium molybdate spectrophotometry; the difference in the sample detection value with and without chloroform added is calibrated with the recovery rate to obtain the phosphorus content of the microorganisms.

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