A method for determining the carbon use efficiency of soil microorganisms

Abstract

The application discloses a kind of determination methods of soil microbial carbon utilization efficiency, it is related to the determination field of carbon utilization efficiency, the application includes pre-culturing soil sample, multi-source 18 O Isotope gradient labeling, dynamic gas monitoring, dynamic correction of microbial respiration rate, microbial growth rate and carbon utilization efficiency acquisition, by multi-source isotope labeling, the contribution of different oxygen sources to microbial DNA synthesis is quantified, the low estimation of carbon utilization efficiency caused by traditional single labeling method is avoided, the limitation problem existing in the current soil microbial carbon utilization efficiency determination process is solved, gradient labeling reduces the inhibitory effect of local concentration on microbial metabolism, improves the uniformity of labeling, sets multiple soil samples, simultaneously uses dynamic monitoring to capture the real-time change of microbial respiration, eliminates the influence of isotope fractionation on carbon dioxide concentration calculation, avoids the instantaneous error of traditional static measurement, to a certain extent, the accuracy of analysis result is improved.

Description

Technical Field

[0001] This application relates to the field of carbon use efficiency measurement technology, specifically to a method for measuring soil microbial carbon use efficiency. Background Technology

[0002] Soil microbial carbon use 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 an important parameter for predicting carbon transport and carbon storage capacity in terrestrial ecosystems.

[0003] Existing measurement techniques mainly employ 13 C or 14 Carbon-labeled substrates are used to extrapolate carbon use efficiency based on changes in microbial biomass, but this method does not consider microbial absorption preferences for different substrates, potentially overestimating carbon use efficiency. In recent years, the use of... 18 A method for tracking microbial growth rates and estimating microbial carbon use efficiency using O-labeled water has been proposed. However, this method assumes that all oxygen in DNA synthesis comes from water, neglecting the contribution of oxygen from the substrate, which may lead to an underestimation of carbon use efficiency. Furthermore, existing methods generally do not consider the oxygen isotope fractionation effect in respiration rate measurements, which can also cause errors in carbon use efficiency estimation. Therefore, this proposal suggests using gradient labeling and dynamic gas analysis to offset the errors from multi-source labeling through a dynamic integration algorithm.

[0004] Existing technology, such as the invention application patent with publication number CN113444612A, discloses a microcosmic culture device and its application in the quantitative analysis of soil carbon diffusion and microbial utilization processes. The microcosmic culture device includes: a sealed container and an incubator and dialysis tube located within the sealed container; the incubator includes a soil layer; the dialysis tube is connected to the incubator, and a portion of the tube extends along its length through the side wall of the incubator into the soil layer; the dialysis tube contains a carbon source, which allows the carbon source to diffuse into the soil layer while maintaining a consistent water potential inside and outside the dialysis tube.

[0005] Regarding the above-mentioned solutions, the inventors of this application have found that the above-mentioned technologies have at least the following technical problems: 1. Currently, there is a lack of multi-isotope labeling such as water, substrate and oxygen, so as to quantify the contribution of different oxygen sources to DNA synthesis. This cannot avoid the underestimation of CUE caused by traditional single labeling. The lack of gradient labeling makes it impossible to reduce the inhibitory effect of excessively high local concentration on microbial metabolism and 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 is not used for respiration rate correction in step four, which cannot eliminate the influence of isotope fractionation on carbon dioxide concentration calculation, thus reducing the accuracy of the analysis results to some 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 soil microbial carbon use efficiency.

[0008] To solve the above-mentioned technical problems, this application adopts the following technical solution: This application provides a method for determining soil microbial carbon use efficiency, including: Step 1, pre-culturing soil samples: the sampled soil is sieved, the soil moisture content is adjusted and pre-cultured, and then the pre-cultured soil sample and pre-culture data are obtained.

[0009] Step 2, Multiple Sources Isotope gradient labeling: Pre-cultured soil samples are grouped, and then the types and abundance of markers for each group are set to obtain complete multi-source data. Soil samples from each group labeled with isotope gradients.

[0010] Step 3: Dynamic Gas Monitoring: This involves monitoring multiple gas sources. Dynamic gas monitoring was performed on each group of soil samples labeled with isotopic gradients to obtain monitoring data for each group of soil samples.

[0011] Step 4: Dynamic Correction of Microbial Respiration Rate: Extract gas temperature, gas pressure, carbon dioxide concentration, and oxygen concentration from each group of soil samples. The abundance was then used to correct the carbon dioxide concentration of each group of soil samples, thereby analyzing the microbial respiration rate of each group of soil samples.

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

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

[0014] Preferably, the process of sieving, adjusting soil moisture content, and pre-culturing the sampled soil to obtain pre-cultured soil samples and pre-culture data includes: passing the sampled soil through a soil sieve with a 2mm aperture, weighing 30g of fresh sieved soil and placing it in a culture bottle, adjusting its moisture content to 60% of field capacity, and placing the soil in a constant temperature incubator for pre-culture for 24 hours. Temperature and humidity of the soil are collected at various sampling time points to obtain pre-cultured soil samples, temperature and humidity at each sampling time point, and the average temperature and average humidity are calculated and recorded as pre-culture data.

[0015] Preferably, the acquisition of multiple sources is completed. The isotope gradient-labeled soil samples consist of: dividing the soil samples into four groups, labeled as each group, and placing them in chromatographic bottles; then setting the markers for each group of soil samples. The marker for the first group of soil samples is... The markers for the second group of soil samples were The markers for the third group of soil samples were In the fourth group of soil samples, naturally abundant ultrapure water was injected, and markers were injected at various injection time points using a microfluidic control device, thereby obtaining complete multi-source data. Soil samples were labeled with isotopic gradients, and the concentrations of various components in each soil sample were measured. 18 The percentage of O atoms exceeds 10%.

[0016] Preferably, the method for completing multi-source Dynamic gas monitoring was performed on various groups of soil samples labeled with isotopic gradients, including: completing multi-source... For each group of soil samples labeled with isotope gradients, record the soil height of each sample within the chromatographic bottle. Place the chromatographic bottle in a headspace vial, seal the bottle opening, and then use an isotope-free chromatographic instrument. Air replaces the gas in the headspace vial; when the headspace vial is empty... Gas replacement was stopped, and formal culture was initiated. A negative control for respiration rate was set up, using three empty headspace bottles for gas replacement. Simultaneously, monitoring intervals were established, and the gas temperature, gas pressure, carbon dioxide concentration, and oxygen concentration of each group of soil samples were monitored according to these intervals. Abundance was collected.

[0017] Preferably, the correction of carbon dioxide concentration for each group of soil samples includes: according to the calculation formula. The corrected carbon dioxide concentrations for each group of soil samples were obtained. ,in This is expressed as the carbon dioxide concentration of each group of soil samples. The pressure is expressed as the gas pressure of each group of soil samples. Expressed as the gas constant, These are the gas temperatures of each group of soil samples. This represents 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: according to the calculation formula The correction factor for carbon dioxide concentration in each group of soil samples was determined, among which... Indicates the oxygen content in the bottle Abundance, This indicates the current training duration. This indicates that the oxygen content in the bottle is... The rate of consumption, This is expressed as the monitoring interval duration. Represented as oxygen The slope value of the effect of abundance change on carbon dioxide concentration.

[0019] Preferably, the analysis yields the microbial respiration rate of each group 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. This represents the volume occupied by the gas in the headspace bottle. Expressed as the dry soil mass of the soil sample. This indicates the training duration.

[0020] Preferably, the analysis to determine the microbial growth rate of each group of soil samples includes: calculating the rate according to the formula... The growth rate of each group of soil samples was determined. ,in This represents the amount of DNA produced in each group of soil samples during the culture process. This is represented by the conversion factor of DNA content in each group of soil samples. A correction factor representing the growth rate.

[0021] According to the calculation formula The conversion factor of DNA content in each group of soil samples was determined. ,in This is expressed as the DNA content of each group of soil samples. It is expressed as the carbon content of microbial biomass.

[0022] According to the calculation formula The correction coefficient for the microbial growth rate was obtained, where This indicates that step two has been completed. During gradient labeling, in the soil Atomic percentage of DNA at the end of culture The slope of the linear relationship between atomic percentages; This indicates that step two is complete. During gradient labeling, in the soil The atomic percentage of DNA at the end of culture The slope of the linear relationship between the atomic percentages.

[0023] Preferably, the DNA yield of each group of soil samples includes: according to the calculation formula The DNA yield of each group of soil samples was determined. ,in Expressed as oxygen mass in soil sample DNA This represents the percentage of oxygen assimilated by microorganisms, and 0.3121 represents the mass percentage of oxygen atoms in DNA. Represented as an environmental weight function, where and These represent the mean temperature and mean humidity values ​​in the pre-culture data, respectively.

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

[0025] The beneficial effects of this application are as follows: 1. This application provides a method for determining soil microbial carbon use efficiency, which involves pre-culturing soil samples and using multiple sources. 18 O isotope gradient labeling, dynamic gas monitoring, dynamic correction of microbial respiration rate, acquisition of microbial growth rate and carbon use efficiency—by labeling multiple isotopes, the contribution of different oxygen sources to microbial DNA synthesis is quantified, avoiding the underestimation of carbon use efficiency caused by traditional single labeling methods. This solves the limitations of current soil microbial carbon use efficiency measurement. Gradient labeling reduces the inhibitory effect of excessively high local concentrations on microbial metabolism and improves the uniformity of labeling. Multiple 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 and avoiding the instantaneous errors of traditional static measurements, thus improving the accuracy of the analysis results to a certain extent.

[0026] 2. This application employs 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 excessively high local concentrations on microbial metabolism and improves the uniformity of labeling.

[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, oxygen isotope abundance data is used for respiration rate correction in step four, eliminating the influence of isotope fractionation on carbon dioxide concentration calculation, which improves the accuracy of the analysis results to a certain extent.

[0028] 4. In this application, the original concentration is converted to the molar concentration under standard conditions to eliminate the influence of temperature and pressure fluctuations, and the concentration of oxygen in the molar concentration is calculated. 18 The rate of change of O abundance over time reflects the isotopic fractionation effect of microbial respiration. When microbial respiration leads to oxygen isotopic fractionation, the oxygen content... 18 O abundance changes dynamically. By calculating the product of fractionation rate and monitoring interval, and the slope of fractionation rate and carbon dioxide concentration, the deviation of carbon dioxide concentration is corrected. The discrete corrected carbon dioxide concentration is made continuous by using the trapezoidal integral method to reduce instantaneous fluctuation interference, thereby reducing the error of respiration rate. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a flowchart illustrating the implementation steps of the method described in this application.

[0031] Figure 2 This is a schematic diagram illustrating the contribution of different oxygen sources to DNA synthesis in the method of this application. Detailed Implementation

[0032] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0033] Please see Figure 1 As shown, this application provides a method for determining soil microbial carbon use efficiency, including: Step 1, pre-culturing soil samples: sieving the sampled soil, adjusting the soil moisture content and pre-culturing, thereby obtaining the pre-cultured soil sample and pre-culture data.

[0034] In one specific embodiment, the process of sieving the sampled soil, adjusting the soil moisture content, and pre-culturing it to obtain pre-cultured soil samples and pre-culture data includes: passing the sampled soil through a soil sieve with a 2mm aperture, weighing 30g of fresh sieved soil and placing it in a culture bottle, adjusting its moisture content to 60% of field capacity, and placing the soil in a constant temperature incubator for pre-culture for 24 hours. Temperature and humidity of the soil are collected at various sampling time points to obtain pre-cultured soil samples, temperature and humidity at each sampling time point, and the average temperature and average humidity are calculated and recorded as pre-culture data.

[0035] It should be noted that the purpose of sieving is to remove impurities such as litter, stones, and roots from the soil; the incubator should be kept at 25°C or the temperature required by the experimental design; and the soil moisture should be 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 time interval between each data collection point is the same.

[0037] It should be noted that the average temperature is the sum of the temperature at the current sampling time and the sum of the temperatures at all sampling times prior to the current sampling time; the average humidity is the sum of the humidity at the current sampling time and the sum of the humidity at all sampling times prior to the current sampling time.

[0038] Step 2, Multiple Sources Isotope gradient labeling: Pre-cultured soil samples are grouped, and then the types and abundance of markers for each group are set to obtain complete multi-source data. Soil samples from each group labeled with isotope gradients.

[0039] In one specific embodiment, the acquisition of multiple sources is completed. The isotope gradient-labeled soil samples consist of: dividing the soil samples into four groups, labeled as each group, and placing them in chromatographic bottles; then setting the markers for each group of soil samples. The marker for the first group of soil samples is... The markers for the second group of soil samples were The markers for the third group of soil samples were In the fourth group of soil samples, naturally abundant ultrapure water was injected, and markers were injected at various injection time points using a microfluidic control device, thereby obtaining complete multi-source data. Soil samples were labeled with isotopic gradients, and the concentrations of various components in each soil sample were measured. 18 The percentage of O atoms exceeds 10%.

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

[0041] It should be noted that during the experiment, samples treated with natural abundance and those treated with labeling should be placed separately.

[0042] It should be noted that multi-source The isotopic gradient was set, and after injecting the same volume of marker, the final... The atomic percentage was gradually increased from the natural abundance of 0.2% to 20%, and the volume of the labeled liquid did not exceed 5% of the field capacity to avoid excessive water 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 infiltration into the soil pores.

[0044] This application employs 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 excessively high local concentrations on microbial metabolism and improves the uniformity of labeling.

[0045] Step 3: Dynamic Gas Monitoring: This involves monitoring multiple gas sources. Dynamic gas monitoring was performed on each group of soil samples labeled with isotopic gradients to obtain monitoring data for each group of soil samples.

[0046] It should be noted that the headspace vial has a capacity of 20 ml.

[0047] In one specific embodiment, the completion of multi-source Dynamic gas monitoring was performed on various groups of soil samples labeled with isotopic gradients, including: completing multi-source... For each group of soil samples labeled with isotope gradients, record the soil height of each sample within the chromatographic bottle. Place the chromatographic bottle in a headspace vial, seal the bottle opening, and then use an isotope-free chromatographic instrument. Air replaces the gas in the headspace vial; when the headspace vial is empty... Gas replacement was stopped, and formal culture was initiated. A negative control for respiration rate was set up, using three empty headspace bottles for gas replacement. Simultaneously, monitoring intervals were established, and the gas temperature, gas pressure, carbon dioxide concentration, and oxygen concentration of each group of soil samples were monitored according to these intervals. Abundance was collected.

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

[0049] This application sets up multiple 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, oxygen isotope abundance data is used for respiration rate correction in step four, eliminating the influence of isotope fractionation on carbon dioxide concentration calculation, which improves the accuracy of the analysis results to a certain extent.

[0050] Step 4: Dynamic Correction of Microbial Respiration Rate: Extract gas temperature, gas pressure, carbon dioxide concentration, and oxygen concentration from each group of soil samples. The abundance was then used to correct the carbon dioxide concentration of each group of soil samples, thereby analyzing the microbial respiration rate of each group of soil samples.

[0051] In one specific embodiment, the correction of the carbon dioxide concentration of each group of soil samples includes: according to the calculation formula. The corrected carbon dioxide concentrations for each group of soil samples were obtained. ,in This is expressed as the carbon dioxide concentration of each group of soil samples. The pressure is expressed as the gas pressure of each group of soil samples. Expressed as the gas constant, These are the gas temperatures of each group of soil samples. This represents 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 one specific embodiment, the correction factor corresponding to the carbon dioxide concentration in each group of soil samples includes: according to the calculation formula The correction factor for carbon dioxide concentration in each group of soil samples was determined, among which... Indicates the oxygen content in the bottle Abundance, This indicates the current training duration. This indicates that the oxygen content in the bottle is... The rate of consumption, This is expressed as the monitoring interval duration. Represented as oxygen The slope value of the effect of abundance change on carbon dioxide concentration.

[0055] In one specific embodiment, the analysis to determine the microbial respiration rate of each group of soil samples includes: 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. This represents the volume occupied by the gas in the headspace bottle. Expressed as the dry soil mass of the soil sample. This indicates the training duration.

[0056] It should be noted that the training duration Molar mass of carbon dioxide The mass of dry soil was obtained using the ideal gas law. Used to standardize respiratory rate.

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

[0058] In one specific embodiment, the analysis to determine the microbial growth rate of each group of soil samples includes: according to a calculation formula. The growth rate of each group of soil samples was determined. ,in This represents the amount of DNA produced in each group of soil samples during the culture process. This is represented by the conversion factor of DNA content in each group of soil samples. A correction factor representing the growth rate.

[0059] According to the calculation formula The conversion factor of DNA content in each group of soil samples was determined. ,in This is expressed as the DNA content of each group of soil samples. It is expressed as the carbon content of microbial biomass.

[0060] According to the calculation formula The correction coefficient for the microbial growth rate was obtained, where This indicates that step two has been completed. During gradient labeling, in the soil Atomic percentage of DNA at the end of culture The slope of the linear relationship between atomic percentages; This indicates that step two is complete. During gradient labeling, in the soil The atomic percentage of DNA at the end of culture The slope of the linear relationship between the atomic percentages.

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

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

[0063] It should be noted that the mass of oxygen in the sample DNA and Abundance was determined using elemental analysis-isotope ratio mass spectrometry. During the measurement process, naturally abundant samples and labeled samples were placed separately, and the detection order followed the principle of "naturally abundant samples first, then labeled samples"; the quality of the dried DNA was accurately recorded, and the DNA samples were analyzed immediately after drying.

[0064] It should be noted that in the sample The percentage of atoms in the DNA of soil samples from the labeled treatment group (groups 1 and 2) and the natural abundance control group (group 4) was significantly higher than that of the control group (group 4). The abundance difference was obtained through calculation.

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

[0066] In one specific embodiment, the DNA yield of each group of soil samples includes: according to the calculation formula The DNA yield of each group of soil samples was determined. ,in Expressed as oxygen mass in soil sample DNA This represents the percentage of oxygen assimilated by microorganisms, and 0.3121 represents the mass percentage of oxygen atoms in DNA. Represented as an environmental weight function, where and These represent the mean temperature and mean humidity values ​​in the pre-culture data, respectively.

[0067] It should be noted that, according to the calculation formula The analysis yields the environmental weighting function, where Represented as a temperature term, for example, when When the exponent term is 1, the temperature weight reaches its maximum value of 0.6; when At that time, the weight decays by 10%; among which Represented as a humidity item, for example when When, the linear term is 0.4; when The weights change linearly. .

[0068] It should be noted that the environmental weighting function quantifies the synergistic effect of temperature and humidity, thereby improving the fit.

[0069] It should be noted that the average temperature and humidity values ​​in the pre-culture data in step one are converted into an environmental weighting function in step five for use in dynamically adjusting DNA production; the microbial respiration rate in step four and the growth rate in step five complete the calculation of microbial carbon utilization efficiency in step six, comprehensively reflecting microbial metabolism.

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

[0071] In one specific embodiment, the analysis to determine the microbial carbon use efficiency of each group of soil samples includes: according to the calculation formula The microbial carbon use efficiency corresponding to each group of soil samples was determined. .

[0072] In this application, the original concentration is converted to the molar concentration under standard conditions to eliminate the influence of temperature and pressure fluctuations, and the concentration of oxygen is calculated. 18 The rate of change of O abundance over time reflects the isotopic fractionation effect of microbial respiration. When microbial respiration leads to oxygen isotopic fractionation, the oxygen content... 18 O abundance changes dynamically. By calculating the product of fractionation rate and monitoring interval, and the slope of fractionation rate and carbon dioxide concentration, the deviation of carbon dioxide concentration is corrected. The discrete corrected carbon dioxide concentration is made continuous by using the trapezoidal integral method to reduce instantaneous fluctuation interference, thereby reducing the error of respiration rate.

[0073] It should be noted that, ;when Carbon consumption during microbial growth is greater than or equal to carbon consumption during microbial respiration.

[0074] This application discloses a method for determining soil microbial carbon use efficiency, relating to the field of carbon use efficiency determination technology. This application includes pre-cultured soil samples and multi-source... 18 O isotope gradient labeling, dynamic gas monitoring, dynamic correction of microbial respiration rate, acquisition of microbial growth rate and carbon use efficiency—by labeling multiple isotopes, the contribution of different oxygen sources to microbial DNA synthesis is quantified, avoiding the underestimation of carbon use efficiency caused by traditional single labeling methods. This solves the limitations of current soil microbial carbon use efficiency measurement. Gradient labeling reduces the inhibitory effect of excessively high local concentrations on microbial metabolism and improves the uniformity of labeling. Multiple 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 and avoiding the instantaneous errors of traditional static measurements, thus improving the accuracy of the analysis results to a certain extent.

[0075] The above content is merely an example and illustration of the concept of this application. Those skilled in the art can make various modifications or additions to the specific embodiments described or use similar methods to replace them, as long as they do not deviate from the inventive concept or exceed the scope defined in this specification, they should all fall within the protection scope of this application.

Claims

1. A method for determining soil microbial carbon use efficiency, characterized in that, include: Step 1: Pre-culture soil samples: The sampled soil is sieved, the soil moisture content is adjusted and pre-cultured to obtain pre-cultured soil samples and pre-culture data; Step 2, Multiple Sources Isotope gradient labeling: Pre-cultured soil samples are grouped, and then the types and abundance of markers for each group are set to obtain complete multi-source data. Soil samples from each group labeled with isotopic gradients; The acquisition of multiple sources Soil samples labeled with isotopic gradients include: Soil samples were divided into four groups, denoted as group A and group B, and placed in chromatographic bottles. A marker was then assigned to each group of soil samples. The marker for the first group of soil samples was... The markers for the second group of soil samples were The markers for the third group of soil samples were In the fourth group of soil samples, naturally abundant ultrapure water was injected, and markers were injected at various injection time points using a microfluidic control device, thereby completing the multi-source sampling. Soil samples were labeled with isotopic gradients, and the concentrations of various components in each soil sample were measured. 18 O atoms account for more than 100% of the total; Step 3: Dynamic Gas Monitoring: This involves monitoring multiple gas sources. Dynamic gas monitoring was performed on each group of soil samples labeled with isotopic gradients to obtain monitoring data for each group of soil samples. Step 4: Dynamic Correction of Microbial Respiration Rate: Extract gas temperature, gas pressure, carbon dioxide concentration, and oxygen concentration from each group of soil samples. The abundance was then used to correct the carbon dioxide concentration of each group of soil samples, thereby analyzing the microbial respiration rate of each group of soil samples. The correction of carbon dioxide concentration for each group of soil samples includes: According to the calculation formula The corrected carbon dioxide concentrations for each group of soil samples were obtained. ,in This is expressed as the carbon dioxide concentration of each group of soil samples. The pressure is expressed as the gas pressure of each group of soil samples. Expressed as the gas constant, These are the gas temperatures of each group of soil samples. This indicates the correction factor corresponding to the carbon dioxide concentration in each group of soil samples; The correction factors corresponding to the carbon dioxide concentration in each group of soil samples include: According to the calculation formula The correction factor for carbon dioxide concentration in each group of soil samples was determined, among which... Indicates the oxygen content in the bottle Abundance, This indicates the current training duration. This indicates that the oxygen content in the bottle is... The rate of consumption, This is expressed as the monitoring interval duration. Represented as oxygen The slope value of the effect of abundance change on carbon dioxide concentration; The analysis yielded the microbial respiration rates of each group 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. This represents the volume occupied by the gas in the headspace bottle. Expressed as the dry soil mass of the soil sample. Indicated as incubation duration; Step 5: Microbial growth rate: Extract pre-culture data, extract oxygen mass and... Atomic percentages were used to analyze and determine the microbial growth rate of each group of soil samples. The analysis yielded the 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 determined. ,in This represents the amount of DNA produced in each group of soil samples during the culture process. This is represented by the conversion factor of DNA content in each group of soil samples. Correction factor representing growth rate; According to the calculation formula The conversion factor of DNA content in each group of soil samples was determined. ,in This is expressed as the DNA content of each group of soil samples. Expressed as microbial biomass carbon content; According to the calculation formula The correction coefficient for the microbial growth rate was obtained, where This indicates that step two has been completed. During gradient labeling, in the soil Atomic percentage of DNA at the end of culture The slope of the linear relationship between atomic percentages; This indicates that step two is complete. During gradient labeling, in the soil The atomic percentage of DNA at the end of culture The slope of the linear relationship between atomic percentages; Step 6: Obtaining carbon use efficiency: Extract the microbial respiration rate and growth rate of each group of soil samples, and then analyze the microbial carbon use efficiency of each group of soil samples.

2. The method for determining soil microbial carbon use efficiency according to claim 1, characterized in that, The process of sieving, adjusting soil moisture content, and pre-culturing the sampled soil to obtain pre-cultured soil samples and pre-culture data includes: The sampled soil was passed through a soil sieve with a 2mm aperture. 30g of fresh sieved soil was placed in a culture bottle and its moisture content was adjusted to 60% of the field capacity. The soil was then placed in a constant temperature incubator for 24 hours for pre-culture. Temperature and humidity of the soil were collected at various time points to obtain the pre-cultured soil samples, the temperature and humidity at each collection time point, and the mean temperature and mean humidity were calculated and recorded as pre-culture data.

3. The method for determining soil microbial carbon use efficiency according to claim 2, characterized in that, The completion of multi-source Dynamic gas monitoring was performed on each group of soil samples labeled with isotopic gradients, including: This will complete the multi-source For each group of soil samples labeled with isotope gradients, the soil height of each sample within the chromatographic bottle was recorded. The chromatographic bottle was then placed in a headspace vial, the bottle opening was sealed, and the chromatography was performed without... Air replaces the gas in the headspace vial; when the headspace vial is empty... Gas replacement was stopped, and formal culture was initiated. A negative control for respiration rate was set up, using three empty headspace bottles for gas replacement. Simultaneously, monitoring intervals were established, and the gas temperature, gas pressure, carbon dioxide concentration, and oxygen concentration of each group of soil samples were monitored according to these intervals. Abundance was collected.

4. The method for determining soil microbial carbon use efficiency according to claim 3, characterized in that, The DNA yield of each group of soil samples includes: According to the calculation formula The DNA yield of each group of soil samples was determined. ,in Expressed as oxygen mass in soil sample DNA This represents the percentage of oxygen assimilated by microorganisms, and 0.3121 represents the mass percentage of oxygen atoms in DNA. Represented as an environmental weight function, where and These represent the mean temperature and mean humidity values ​​in the pre-culture data, respectively.

5. The method for determining soil microbial carbon use efficiency according to claim 4, characterized in that, The analysis yielded the microbial carbon use efficiency of each group of soil samples, including: According to the calculation formula The microbial carbon use efficiency corresponding to each group of soil samples was determined. .

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