Method for measuring carbon utilization efficiency of soil microorganisms

Abstract

The invention discloses a soil microorganism carbon utilization efficiency determination method, and relates to the technical field of carbon utilization efficiency determination, and the method comprises soil sample pre-culture, multi-source 18O isotope gradient labeling, dynamic gas monitoring, microorganism respiration rate dynamic correction, and microorganism growth rate and carbon utilization efficiency acquisition. By labeling multi-source isotopes and quantifying contributions of different oxygen sources to microorganism DNA synthesis, underestimation of the carbon utilization efficiency caused by a traditional single labeling method is avoided, the problem of limitation existing in the current soil microorganism carbon utilization efficiency determination process is solved, the inhibition effect of too high local concentration on microorganism metabolism is reduced through gradient labeling, and the accuracy of soil microorganism carbon utilization efficiency determination is improved. The method has the advantages that marking uniformity is improved, multiple groups of soil samples are arranged, real-time change of microbial respiration is captured by means of dynamic monitoring, influence of isotope fractionation on carbon dioxide concentration calculation is eliminated, instantaneous errors of traditional static measurement are avoided, and accuracy of analysis results is improved to a certain extent.

Description

Technical Field

[0001] The present application relates to the technical field of carbon utilization efficiency determination, and in particular to a method for determining soil microbial carbon utilization efficiency. Background Art

[0002] Soil microbial carbon utilization efficiency, defined as the ratio of carbon allocated to growth by microorganisms to the total carbon absorbed, is a key indicator for measuring microbial anabolism and catabolism, and is also an important parameter for predicting carbon flow and storage capacity of terrestrial ecosystems.

[0003] The existing measurement technology mainly uses 13 C or 14 C-labeled substrates are used to estimate carbon utilization efficiency in combination with changes in microbial biomass, but the absorption preference of microorganisms for different substrates is not considered, which may overestimate carbon utilization efficiency. 18 A method has been proposed to track microbial growth rates and estimate carbon utilization efficiency using O-labeled water. However, this method assumes that all oxygen in DNA synthesis comes from water, neglecting the contribution of oxygen in the substrate, which can lead to an underestimation of carbon utilization efficiency. Furthermore, existing methods generally fail to account for oxygen isotope fractionation in respiration rate measurements, which can also lead to biased carbon utilization efficiency estimates. Therefore, this approach proposes combining gradient labeling with dynamic gas analysis to offset the errors from multi-source labeling using a dynamic integration algorithm.

[0004] Prior art, such as the invention patent application with publication number CN113444612A, discloses a microcosm cultivation device and its application in the quantitative analysis of soil carbon diffusion and microbial utilization. The microcosm cultivation device comprises a sealed container, an incubator and a dialysis tube within the sealed container; the incubator includes a soil layer; the dialysis tube is connected to the incubator, with a portion of the tube extending longitudinally through the side wall of the incubator into the soil layer. The dialysis tube contains a carbon source, which diffuses the carbon source into the soil layer while maintaining a consistent water potential inside and outside the dialysis tube.

[0005] Regarding the above scheme, the inventors of this application found that the above technology has at least the following technical problems: 1. There is currently a lack of multi-isotope labels such as water, substrate and oxygen to quantify the contribution of different oxygen sources to DNA synthesis. It cannot avoid the underestimation of CUE caused by traditional single labeling. There is a lack of gradient labeling, which cannot reduce the inhibitory effect of local high concentration on microbial metabolism and cannot improve the uniformity of labeling.

[0006] 2. Currently, dynamic monitoring is not used to capture real-time changes in microbial respiration, which cannot avoid the instantaneous errors of traditional static measurements. At the same time, oxygen isotope abundance data are not used for respiration rate correction in step 4, which cannot eliminate the impact of isotope fractionation on carbon dioxide concentration calculations, thereby reducing the accuracy of the analysis results to a certain extent. Summary of the Invention

[0007] In view of the above-mentioned technical deficiencies, the purpose of this application is to provide a method for determining the carbon utilization efficiency of soil microorganisms.

[0008] In order to solve the above technical problems, the present application adopts the following technical solution: The present application provides a method for determining the carbon utilization efficiency of soil microorganisms, including: Step 1, pre-cultivating soil samples: screening the sampled soil, adjusting the soil moisture content and pre-cultivating, and then obtaining the pre-cultivated soil samples and pre-cultivation data.

[0009] Step 2: Multiple Sources Isotope gradient labeling: The soil samples after pre-cultivation are grouped, and the marker types and marker abundances of each group of soil samples are set to obtain multi-source Soil samples from various groups were labeled with isotope gradients.

[0010] Step 3: Dynamic gas monitoring: Complete multi-source Dynamic gas monitoring is performed on each group of soil samples labeled with isotope gradients to obtain monitoring data for each group of soil samples.

[0011] Step 4: Dynamic correction of microbial respiration rate: Extract the gas temperature, gas pressure, carbon dioxide concentration and oxygen content of each soil sample. The abundance of soil microorganisms was determined by analyzing the carbon dioxide concentration of each soil sample to obtain the microbial respiration rate of each soil sample.

[0012] Step 5. Microbial growth rate: Extract pre-culture data, extract oxygen mass and The atomic percentage was exceeded, and the microbial growth rate of each group of soil samples was analyzed.

[0013] Step 6: Obtaining carbon utilization efficiency: Extract the microbial respiration rate and growth rate of each group of soil samples, and then analyze and obtain the microbial carbon utilization efficiency of each group of soil samples.

[0014] Preferably, the sieving of the sampled soil, adjusting the soil moisture content and pre-culturing, and then obtaining the soil sample and pre-culturing data after pre-culturing, comprises: passing the sampled soil through a soil sieve with a pore size of 2 mm, weighing 30 g of freshly sieved soil and placing it in a culture bottle, adjusting its moisture content to 60% of the field water holding capacity, and placing the soil in a constant temperature box for pre-culturing for 24 hours, and setting each collection time point to collect the temperature and humidity of the soil, thereby obtaining the soil sample after pre-culturing, the temperature and humidity corresponding to each collection time point, respectively calculating the temperature mean and humidity mean, and recording them as pre-culturing data.

[0015] Preferably, the acquisition is completed from multiple sources The isotope gradient labeled soil samples include: dividing the soil samples into four groups, each group of soil samples, and placing them into chromatographic bottles, and then setting the markers for each group of soil samples. The marker of the first group of soil samples is , the markers of the second group of soil samples were , the markers of the third group of soil samples are The fourth group of soil samples was injected with ultrapure water of natural abundance, and markers were injected at each injection time point through a microfluidic control device to obtain the multi-source The soil samples of each group were labeled with isotope gradients, and the 18 The atomic percentage of O exceeds .

[0016] Preferably, the pair completes multiple sources Dynamic gas monitoring of each group of soil samples labeled with isotope gradients, including: Each group of soil samples labeled with isotope gradients, record the soil height of each group of samples in the chromatographic bottle, place the chromatographic bottle in the headspace bottle, seal the bottle mouth and use Air replaces the gas in the headspace bottle. Stop the replacement when the temperature is too low and start formal cultivation; set a negative control for respiration rate and use three empty headspace bottles for gas replacement; set the monitoring interval time at the same time, and measure the gas temperature, gas pressure, carbon dioxide concentration and oxygen content of each group of soil samples according to the monitoring interval time. Abundance is collected.

[0017] Preferably, the correction of the carbon dioxide concentration of each group of soil samples includes: according to the calculation formula Obtain the corrected carbon dioxide concentration of each group of soil samples ,in Expressed as the carbon dioxide concentration of each group of soil samples, Expressed as the gas pressure of each group of soil samples, Expressed as the gas constant, is the gas temperature of each group of soil samples, Indicates the correction factor corresponding to the carbon dioxide concentration in each group of soil samples.

[0018] Preferably, the correction factor corresponding to the carbon dioxide concentration in each group of soil samples includes: The CO2 concentration correction factor in each group of soil samples was obtained, where Indicates the oxygen in the bottle abundance, Indicates the current training time. It means that the oxygen in the bottle The consumption rate, Indicates the monitoring interval duration, Expressed as oxygen Slope value of the effect of abundance changes on CO2 concentration.

[0019] Preferably, the analysis to obtain the microbial respiration rate of each group of soil samples includes: calculating the microbial respiration rate according to the formula The microbial respiration rate of each group of soil samples was obtained , Expressed as the molar mass of carbon dioxide carbon, It is expressed as the volume occupied by the gas in the headspace bottle. Expressed as the dry mass of the soil sample, Indicated as the cultivation time.

[0020] Preferably, the analysis to obtain the microbial growth rate of each group of soil samples includes: calculating the growth rate of microorganisms according to the formula The growth rate of each group of soil samples was obtained ,in It is expressed as the DNA output of each soil sample during the cultivation process. Expressed as the conversion factor for the DNA content in each group of soil samples, represents the correction factor for growth rate.

[0021] According to the calculation formula Obtain the conversion factor of DNA content in each group of soil samples ,in Expressed as the DNA content of each soil sample, Expressed as microbial biomass carbon content.

[0022] According to the calculation formula The correction factor for microbial growth rate is obtained, where Indicates that step 2 is completed Gradient marking, soil The atomic percentage of DNA at the end of culture The slope of the linear relationship between the atomic percentage excess of ; Indicates completion of step 2 Gradient marking, soil The atomic percentage of DNA at the end of the culture The slope of the linear relationship between the atomic percentage excess.

[0023] Preferably, the DNA yield of each group of soil samples includes: Determine the DNA yield of each soil sample ,in Expressed as the amount of oxygen in the DNA of the soil sample It is expressed as the proportion of labeled oxygen assimilated by microorganisms, and 0.3121 is expressed as the mass proportion of oxygen atoms in DNA. Expressed as an environmental weight function, where and Represented as the mean temperature and mean humidity in the pre-cultivation data, respectively.

[0024] Preferably, the analysis to obtain the microbial carbon utilization efficiency of each group of soil samples includes: calculating the carbon utilization efficiency of each group of soil samples according to the formula The microbial carbon utilization efficiency corresponding to each group of soil samples was obtained .

[0025] The beneficial effects of the present application are as follows: 1. The present application provides a method for determining the carbon utilization efficiency of soil microorganisms, which can be achieved by pre-culturing soil samples, multi-source 18 O isotope gradient labeling, dynamic gas monitoring, dynamic correction of microbial respiration rate, acquisition of microbial growth rate and carbon utilization efficiency. By labeling multiple isotopes, the contribution of different oxygen sources to microbial DNA synthesis is quantified, avoiding the underestimated carbon utilization efficiency caused by traditional single labeling methods, and solving the limitations of the current soil microbial carbon utilization efficiency determination process. Gradient labeling reduces the inhibitory effect of local excessive concentration on microbial metabolism and improves the uniformity of labeling. Multiple groups of soil samples are set up, and dynamic monitoring is used to capture real-time changes in microbial respiration, eliminating the influence of isotope fractionation on carbon dioxide concentration calculation, avoiding the instantaneous error of traditional static measurement, and improving the accuracy of the analysis results to a certain extent.

[0026] 2. This application uses multi-isotope labeling, such as water, substrate, and oxygen, to quantify the contribution of different oxygen sources to DNA synthesis, avoiding the underestimation of CUE caused by traditional single labeling; gradient labeling reduces the inhibitory effect of local high concentration on microbial metabolism and improves labeling uniformity.

[0027] 3. This application sets up multiple groups of soil samples and uses dynamic monitoring to capture real-time changes in microbial respiration, avoiding the instantaneous errors of traditional static measurements; at the same time, the oxygen isotope abundance data is used to correct the respiration rate in step 4 to eliminate the influence of isotope fractionation on the calculation of carbon dioxide concentration, thereby improving the accuracy of the analysis results to a certain extent.

[0028] 4. In this application, the original concentration is converted into the molar concentration under standard conditions to eliminate the influence of temperature and pressure fluctuations and calculate the oxygen content. 18 The rate of change of O abundance over time reflects the isotope fractionation effect of microbial respiration. When microbial respiration causes oxygen isotope fractionation, the oxygen 18 O abundance changes dynamically. The deviation of carbon dioxide concentration is corrected by calculating the product of the fractionation rate and the monitoring interval, and the slope value of the fractionation rate and carbon dioxide concentration. The discrete corrected carbon dioxide concentration is continuousized using the trapezoidal integration method to reduce the interference of instantaneous fluctuations and thus reduce the error of respiratory rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0030] Figure 1 This is a flowchart of the steps for implementing the application method.

[0031] Figure 2 Schematic diagram of the contribution of different oxygen sources to DNA synthesis in the method of this application. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0033] See also Figure 1 As shown, the present application provides a method for determining the carbon utilization efficiency of soil microorganisms, including: step 1, pre-culturing soil samples: screening the sampled soil, adjusting the soil moisture content and pre-culturing, and then obtaining the pre-cultured soil samples and pre-culture data.

[0034] In a specific embodiment, the sieving of the sampled soil, adjusting the soil moisture content and pre-culturing, and then obtaining the pre-cultured soil sample and pre-culture data, includes: passing the sampled soil through a soil sieve with a pore size of 2 mm, weighing 30 g of freshly sieved soil and placing it in a culture bottle, adjusting its moisture content to 60% of the field water holding capacity, and placing the soil in a constant temperature box for pre-culture for 24 hours, and setting each collection time point to collect the temperature and humidity of the soil, thereby obtaining the pre-cultured soil sample, the temperature and humidity corresponding to each collection time point, respectively calculating the temperature mean and humidity mean, and recording them as pre-culture data.

[0035] It should be noted that the purpose of screening is to remove impurities such as litter, stones and roots in the soil; the incubator is maintained at 25°C or the temperature required by the experimental design; the soil moisture is kept constant during the pre-culture period to ensure that the microorganisms are in a stable metabolic state.

[0036] It should be noted that the intervals between the various collection time points are the same.

[0037] It should be noted that the mean temperature is expressed as the average of the temperature value at the current collection time point and the temperature at each collection time point before the current collection time point; the mean humidity is expressed as the average of the humidity value at the current collection time point and the humidity at each collection time point before the current collection time point.

[0038] Step 2: Multiple Sources Isotope gradient labeling: The soil samples after pre-cultivation are grouped, and the marker types and marker abundances of each group of soil samples are set to obtain multi-source Soil samples from various groups were labeled with isotope gradients.

[0039] In a specific embodiment, the acquisition is completed from multiple sources The isotope gradient labeled soil samples include: dividing the soil samples into four groups, each group of soil samples, and placing them into chromatographic bottles, and then setting the markers for each group of soil samples. The marker of the first group of soil samples is , the markers of the second group of soil samples were , the markers of the third group of soil samples are The fourth group of soil samples was injected with ultrapure water of natural abundance, and markers were injected at each injection time point through a microfluidic control device to obtain the multi-source The soil samples of each group were labeled with isotope gradients, and the 18 The atomic percentage of O exceeds .

[0040] It should be noted that the soil samples were divided into four groups, each of which was For each abundance treatment, 1 g of fresh soil was weighed.

[0041] It should be noted that during the experiment, care should be taken to separate the natural abundance treatment and labeled treatment samples.

[0042] It should be noted that multiple sources The isotope gradient was set up so that the final concentrations of the first, second and third soil samples were the same after injecting the same volume of marker. The atomic percentage is gradually increased from 0.2% of the natural abundance to 20%, and the volume of the marker liquid does not exceed 5% of the field water holding capacity to avoid excessive moisture inhibiting microbial metabolism.

[0043] It should be noted that the microfluidic control device injects the marker at a set flow rate, and microscopic observation is used to ensure uniform penetration of soil pores.

[0044] This application uses multi-isotope labeling such as water, substrate, and oxygen to quantify the contribution of different oxygen sources to DNA synthesis, avoiding the underestimation of CUE caused by traditional single labeling; gradient labeling reduces the inhibitory effect of local excessive concentration on microbial metabolism and improves the uniformity of labeling.

[0045] Step 3: Dynamic gas monitoring: Complete multi-source Dynamic gas monitoring is performed on each group of soil samples labeled with isotope gradients to obtain monitoring data for each group of soil samples.

[0046] It should be noted that the capacity of the headspace bottle is 20 ml.

[0047] In one embodiment, the multi-source Dynamic gas monitoring of each group of soil samples labeled with isotope gradients, including: Each group of soil samples labeled with isotope gradients, record the soil height of each group of samples in the chromatographic bottle, place the chromatographic bottle in the headspace bottle, seal the bottle mouth and use Air replaces the gas in the headspace bottle. Stop the replacement when the temperature is too low and start formal cultivation; set a negative control for respiration rate and use three empty headspace bottles for gas replacement; set the monitoring interval time at the same time, and measure the gas temperature, gas pressure, carbon dioxide concentration and oxygen content of each group of soil samples according to the monitoring interval time. Abundance is collected.

[0048] It should be noted that the gas temperature is obtained by a temperature sensor, and the carbon dioxide concentration is obtained by a micro gas sensor.

[0049] This application sets up multiple groups of soil samples and uses dynamic monitoring to capture real-time changes in microbial respiration, avoiding the instantaneous errors of traditional static measurements; at the same time, the oxygen isotope abundance data is used to correct the respiration rate in step four, eliminating the influence of isotope fractionation on the calculation of carbon dioxide concentration, and to a certain extent improving the accuracy of the analysis results.

[0050] Step 4: Dynamic correction of microbial respiration rate: Extract the gas temperature, gas pressure, carbon dioxide concentration and oxygen content of each soil sample. The abundance of soil microorganisms was determined by analyzing the carbon dioxide concentration of each soil sample to obtain the microbial respiration rate of each soil sample.

[0051] In a specific embodiment, the correction of the carbon dioxide concentration of each group of soil samples includes: according to the calculation formula Obtain the corrected carbon dioxide concentration of each group of soil samples ,in Expressed as the carbon dioxide concentration of each group of soil samples, Expressed as the gas pressure of each group of soil samples, Expressed as the gas constant, is the gas temperature of each group of soil samples, Indicates the correction factor corresponding to the carbon dioxide concentration in each group of soil samples.

[0052] It should be noted that the gas constant .

[0053] It should be noted that the gas pressure is obtained by a pressure sensor.

[0054] In a specific embodiment, the correction factor corresponding to the carbon dioxide concentration in each group of soil samples includes: The CO2 concentration correction factor in each group of soil samples was obtained, where Indicates the oxygen in the bottle abundance, Indicates the current training time. It means that the oxygen in the bottle The consumption rate, Indicates the monitoring interval duration, Expressed as oxygen Slope value of the effect of abundance changes on CO2 concentration.

[0055] In a specific embodiment, the analysis to obtain the microbial respiration rate of each group of soil samples includes: calculating the microbial respiration rate according to the formula The microbial respiration rate of each group of soil samples was obtained , Expressed as the molar mass of carbon dioxide carbon, It is expressed as the volume occupied by the gas in the headspace bottle. Expressed as the dry mass of the soil sample, Indicated as the cultivation time.

[0056] It should be noted that the training time , the molar mass of carbon dioxide The ideal gas law is used to obtain the dry soil mass. Used to normalize respiratory rate.

[0057] Step 5. Microbial growth rate: Extract pre-culture data, extract oxygen mass and The atomic percentage was exceeded, and the microbial growth rate of each group of soil samples was analyzed.

[0058] In a specific embodiment, the analysis to obtain the microbial growth rate of each group of soil samples includes: according to the calculation formula The growth rate of each group of soil samples was obtained ,in It is expressed as the DNA output of each soil sample during the cultivation process. Expressed as the conversion factor for the DNA content in each group of soil samples, represents the correction factor for growth rate.

[0059] According to the calculation formula Obtain the conversion factor of DNA content in each group of soil samples ,in Expressed as the DNA content of each group of soil samples, Expressed as microbial biomass carbon content.

[0060] According to the calculation formula The correction factor for microbial growth rate is obtained, where Indicates that step 2 is completed Gradient marking, soil The atomic percentage of DNA at the end of culture The slope of the linear relationship between the atomic percentage excess of ; Indicates completion of step 2 Gradient marking, soil The atomic percentage of DNA at the end of the culture The slope of the linear relationship between the atomic percentage excess.

[0061] It should be noted that the and All by Figure 2 The linear regression analysis shown was obtained to correct for the effects of different oxygen sources on DNA synthesis.

[0062] It should be noted that the microbial DNA in the soil samples was extracted using the Powersoil DNA kit, and the DNA should be retained to the greatest extent during the extraction process.

[0063] It should be noted that the oxygen mass in the sample DNA and Abundance was determined using elemental analysis coupled with isotope ratio mass spectrometry. During the measurement process, naturally abundant samples and labeled samples were kept separate, and the testing order followed the principle of "natural abundance samples first, then labeled samples." The mass of the dried DNA was accurately recorded, and DNA samples were tested immediately after drying.

[0064] It should be noted that the samples The atomic percentage of the soil samples in the labeled treatment groups (groups 1 and 2) and the natural abundance control group (group 4) was The abundance difference was calculated.

[0065] It should be noted that the MBC and DNA content data in the formula need to come from the same soil sample to ensure a one-to-one correspondence and reduce errors caused by sample inconsistency.

[0066] In a specific embodiment, the DNA yield of each group of soil samples includes: Determine the DNA yield of each soil sample ,in Expressed as the amount of oxygen in the DNA of the soil sample It is expressed as the proportion of labeled oxygen assimilated by microorganisms, and 0.3121 is expressed as the mass proportion of oxygen atoms in DNA. Expressed as an environmental weight function, where and Represented as the mean temperature and mean humidity in the pre-cultivation data, respectively.

[0067] It should be noted that according to the calculation formula The environmental weight function is obtained by analysis, where Expressed as a temperature term, for example, when When the exponential term is 1, the temperature weight reaches a maximum value of 0.6; when When , the weight decays by 10%; Expressed as a humidity term, for example when When , the linear term is 0.4; when , the weight changes linearly .

[0068] It should be noted that the environmental weight function quantifies the synergistic effect of temperature and humidity to improve the fitting.

[0069] It should be noted that the mean temperature and humidity values ​​in the pre-culture data in step one are converted into environmental weight functions in step five for use in dynamically adjusting DNA yield; the microbial respiration rate in step four and the growth rate in step five are used to complete the microbial carbon utilization efficiency determination and calculation in step six, comprehensively reflecting microbial metabolism.

[0070] Step 6: Obtaining carbon utilization efficiency: Extract the microbial respiration rate and growth rate of each group of soil samples, and then analyze and obtain the microbial carbon utilization efficiency of each group of soil samples.

[0071] In a specific embodiment, the analysis to obtain the microbial carbon utilization efficiency of each group of soil samples includes: calculating the carbon utilization efficiency of each group of soil samples according to the formula The microbial carbon utilization efficiency corresponding to each group of soil samples was obtained .

[0072] In this application, the original concentration is converted into the molar concentration under standard conditions to eliminate the influence of temperature and pressure fluctuations and calculate the oxygen content. 18 The rate of change of O abundance over time reflects the isotope fractionation effect of microbial respiration. When microbial respiration causes oxygen isotope fractionation, the oxygen 18 O abundance changes dynamically. The deviation of carbon dioxide concentration is corrected by calculating the product of the fractionation rate and the monitoring interval, and the slope value of the fractionation rate and carbon dioxide concentration. The discrete corrected carbon dioxide concentration is continuousized using the trapezoidal integration method to reduce the interference of instantaneous fluctuations and thus reduce the error of respiratory rate.

[0073] It should be noted that ;when When the carbon consumption in the growth process of microorganisms is greater than or equal to the consumption in microbial respiration.

[0074] This application discloses a method for determining the carbon utilization efficiency of soil microorganisms, which relates to the technical field of carbon utilization efficiency determination. This application includes pre-cultured soil samples, multi-source 18 O isotope gradient labeling, dynamic gas monitoring, dynamic correction of microbial respiration rate, acquisition of microbial growth rate and carbon utilization efficiency. By labeling multiple isotopes, the contribution of different oxygen sources to microbial DNA synthesis is quantified, avoiding the underestimated carbon utilization efficiency caused by traditional single labeling methods, and solving the limitations of the current soil microbial carbon utilization efficiency determination process. Gradient labeling reduces the inhibitory effect of local excessive concentration on microbial metabolism and improves the uniformity of labeling. Multiple groups of soil samples are set up, and dynamic monitoring is used to capture real-time changes in microbial respiration, eliminating the influence of isotope fractionation on carbon dioxide concentration calculation, avoiding the instantaneous error of traditional static measurement, and improving the accuracy of the analysis results to a certain extent.

[0075] The above content is merely an example and explanation of the concept of the present application. Technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined in this specification, they should all fall within the scope of protection of the present application.

Claims

1. A method for determining soil microbial carbon utilization efficiency, characterized in that: include: Step 1: Pre-cultivating soil samples: Screening the sampled soil, adjusting the soil moisture content, and pre-cultivating it, thereby obtaining pre-cultivated soil samples and pre-cultivation data; Step 2: Multiple Sources Isotope gradient labeling: The soil samples after pre-cultivation are grouped, and the marker types and marker abundances of each group of soil samples are set to obtain multi-source Each group of soil samples labeled with isotope gradients; Step 3: Dynamic gas monitoring: Complete multi-source Dynamic gas monitoring is performed on each group of soil samples labeled with isotope gradients to obtain monitoring data for each group of soil samples; Step 4: Dynamic correction of microbial respiration rate: Extract the gas temperature, gas pressure, carbon dioxide concentration and oxygen content of each soil sample. The abundance of soil samples was then corrected for the carbon dioxide concentration of each group of soil samples, and the microbial respiration rate of each group of soil samples was analyzed. Step 5. Microbial growth rate: Extract pre-culture data, extract oxygen mass and The atomic percentage was exceeded, and the microbial growth rate of each group of soil samples was analyzed; Step 6: Obtaining carbon utilization efficiency: Extract the microbial respiration rate and growth rate of each group of soil samples, and then analyze and obtain the microbial carbon utilization efficiency of each group of soil samples.

2. The method for determining soil microbial carbon utilization efficiency according to claim 1, wherein: The method of screening the sampled soil, adjusting the soil moisture content, and pre-culturing the soil to obtain the pre-cultured soil sample and pre-culture data includes: The sampled soil was passed through a soil sieve with a pore size of 2 mm. 30 g of freshly sieved soil was weighed and placed in a culture bottle. The moisture content was adjusted to 60% of the field water holding capacity. The soil was placed in a constant temperature box for pre-culture for 24 hours, and the temperature and humidity of the soil were collected at each collection time point. The soil samples after pre-culture, the temperature and humidity corresponding to each collection time point were obtained, the mean temperature and mean humidity were calculated, and recorded as pre-culture data.

3. The method for determining soil microbial carbon utilization efficiency according to claim 2, wherein: The acquisition is completed from multiple sources The soil samples were labeled with isotope gradients, including: The soil samples were divided into four groups, each group of soil samples was recorded, and placed in a chromatographic bottle. Then, the markers of each group of soil samples were set. The marker of the first group of soil samples was , the markers of the second group of soil samples were , the markers of the third group of soil samples are The fourth group of soil samples was injected with ultrapure water of natural abundance, and markers were injected at each injection time point through a microfluidic control device to obtain the multi-source The soil samples of each group were labeled with isotope gradients, and the The atomic percentage exceeds .

4. The method for determining soil microbial carbon utilization efficiency according to claim 3, wherein: The pair completes the multi-source Dynamic gas monitoring of each set of soil samples labeled with isotope gradients includes: Will complete the multi-source Each group of soil samples labeled with isotope gradients, record the soil height of each group of samples in the chromatographic bottle, place the chromatographic bottle in the headspace bottle, seal the bottle mouth and use Air replaces the gas in the headspace bottle. Stop the replacement when the temperature is too low and start formal cultivation; set a negative control for respiration rate and use three empty headspace bottles for gas replacement; set the monitoring interval time at the same time, and measure the gas temperature, gas pressure, carbon dioxide concentration and oxygen content of each group of soil samples according to the monitoring interval time. Abundance is collected.

5. The method for determining soil microbial carbon utilization efficiency according to claim 4, wherein: The correction of the carbon dioxide concentration of each group of soil samples includes: According to the calculation formula Obtain the corrected carbon dioxide concentration of each group of soil samples ,in Expressed as the carbon dioxide concentration of each group of soil samples, Expressed as the gas pressure of each group of soil samples, Expressed as the gas constant, is the gas temperature of each group of soil samples, Indicates the correction factor corresponding to the carbon dioxide concentration in each group of soil samples.

6. The method for determining soil microbial carbon utilization efficiency according to claim 5, wherein: The correction factors corresponding to the carbon dioxide concentration in each group of soil samples include: According to the calculation formula The CO2 concentration correction factor in each group of soil samples was obtained, where Indicates the oxygen in the bottle abundance, Indicates the current training time. It means that the oxygen in the bottle The consumption rate, Indicates the monitoring interval duration, Expressed as oxygen Slope value of the effect of abundance changes on CO2 concentration.

7. The method for determining soil microbial carbon utilization efficiency according to claim 6, wherein: The analysis yielded microbial respiration rates for each set of soil samples, including: According to the calculation formula The microbial respiration rate of each group of soil samples was obtained , Expressed as the molar mass of carbon dioxide carbon, It is expressed as the volume occupied by the gas in the headspace bottle. Expressed as the dry mass of the soil sample, Indicated as the cultivation time.

8. The method for determining soil microbial carbon utilization efficiency according to claim 7, wherein: The analysis yielded microbial growth rates for each group of soil samples, including: According to the calculation formula The growth rate of each group of soil samples was obtained ,in It is expressed as the DNA output of each soil sample during the cultivation process. Expressed as the conversion factor for the DNA content in each group of soil samples, represents the correction factor for growth rate; According to the calculation formula Obtain the conversion factor of DNA content in each group of soil samples ,in Expressed as the DNA content of each group of soil samples, Expressed as microbial biomass carbon content; According to the calculation formula The correction factor for microbial growth rate is obtained, where Indicates that step 2 is completed Gradient marking, soil The atomic percentage of DNA at the end of culture The slope of the linear relationship between the atomic percentage excess of ; Indicates completion of step 2 Gradient marking, soil The atomic percentage of DNA at the end of the culture The slope of the linear relationship between the atomic percentage excess.

9. The method for determining soil microbial carbon utilization efficiency according to claim 8, wherein: The DNA yield of each soil sample group includes: According to the calculation formula Determine the DNA yield of each soil sample ,in Expressed as the amount of oxygen in the DNA of the soil sample It is expressed as the proportion of labeled oxygen assimilated by microorganisms, and 0.3121 is expressed as the mass proportion of oxygen atoms in DNA. Expressed as an environmental weight function, where and Represented as the mean temperature and mean humidity in the pre-cultivation data, respectively.

10. The method for determining soil microbial carbon utilization efficiency according to claim 9, wherein: The analysis yielded the microbial carbon utilization efficiency of each soil sample group, including: According to the calculation formula The microbial carbon utilization efficiency corresponding to each group of soil samples was obtained .

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