A method for quantifying microbial activity based on stable isotope tracing

Abstract

This application discloses a method for quantifying microbial activity based on stable isotope tracing, belonging to the field of stable isotope tracing and microbial process technology. The method includes: obtaining the optimal culture environment for potential microorganisms within the target microorganism; setting up multiple groups of optimal culture environments corresponding to the potential microorganisms, with different proportions of additives in each group of culture environments. 18 A mixture of O-H2O and naturally abundant water was prepared; potential microorganisms were cultured, and the critical threshold for the response of each potential microorganism was obtained; the abundance of stable isotope labeling was determined, and the isotope abundance curves for each potential microorganism were obtained; environmental samples were pretreated to obtain the samples to be measured; the samples to be measured were placed in all optimal culture environments, and the results were obtained in different optimal culture environments. 18 O isotope incorporation information; based on target microbial groups 18 The degree of O isotope incorporation is used to obtain the activity response results of target microbial groups. This improves the accuracy and reliability of stable isotope probe technology in microbial activity analysis.

Description

Technical Field

[0001] This application belongs to the field of stable isotope tracing and microbial process technology, specifically, it relates to a method for quantifying microbial activity based on stable isotope tracing. Background Technology

[0002] Stable isotope tracing techniques, especially those based on 18 O-enriched water ( 18 Stable isotope probing (SIP) of O-H2O is a commonly used technique for analyzing microbial growth processes. This method involves... 18 The introduction of oxygen isotopes into the reaction system allows for incorporation into the nucleic acid molecules of microorganisms during growth, thus characterizing the microbial growth process. In SIP methods, isotope labeling abundance is a key technical parameter, typically predetermined by experimental conditions. Current techniques often employ fixed isotope abundances for labeling, lacking a systematic evaluation and optimization mechanism for changes in microbial responses under different isotope abundances. Microbial responses may change under different isotope abundances, especially at higher abundances, potentially affecting the reliability of microbial growth and related measurement results obtained using SIP methods. Furthermore, current stable isotope probe techniques primarily focus on obtaining microbial isotope labeling results, lacking further analysis and application based on differences in microbial responses under different isotope enrichment conditions. Especially when different microbial groups exhibit differentiated responses to isotope enrichment levels, current techniques lack a technical solution for dynamically optimizing stable isotope labeling abundance and assessing community activity responses based on microbial response thresholds. This limits the application effectiveness and widespread adoption of stable isotope probe techniques in complex environmental samples and microbial community activity analysis.

[0003] Therefore, existing technologies lack a technical means to optimize or correct the abundance of isotope labels in the SIP method, as well as a basis for determining the abundance of isotope labels based on changes in microbial response and a technical solution for determining microbial taxa based on this technology. There is an urgent need for a solution that can reasonably optimize or limit the abundance of isotope labels while ensuring the effectiveness of the SIP method and directly apply the technical effects. To solve the above problems, this invention proposes a method for optimizing the abundance of stable isotope probe labels. Summary of the Invention

[0004] To address the challenges of guaranteeing the effectiveness of existing SIP methods and the difficulty in applying effective SIP methods to microbial population detection, this application discloses a method for quantifying microbial activity based on stable isotope tracing, specifically:

[0005] A method for quantifying microbial activity based on stable isotope tracing, the determination method comprising:

[0006] Based on the microbial group of the test object, obtain the optimal culture environment for potential microorganisms within the test object;

[0007] Multiple optimal culture environments were set up for potential microorganisms, with different proportions of [unspecified ingredient] added to each culture environment. 18 A mixture of O-H2O and naturally abundant water was used to obtain an isotopic culture environment for potential microorganisms.

[0008] Potential microorganisms were cultured in an isotopic culture environment group to obtain the critical threshold of each potential microorganism's response.

[0009] The abundance of stable isotope markers was determined based on the critical threshold of potential microbial responses, and the abundance of each potential microorganism in its optimal culture environment was obtained. 18 O isotope abundance curve;

[0010] Environmental samples are pretreated to obtain the samples to be tested;

[0011] The samples to be tested were placed in all the optimal culture environments with stable isotope labeling, and the results were obtained in different optimal culture environments. 18 O isotope incorporation information;

[0012] Based on target microbial groups 18 The degree of O isotope incorporation was used to obtain the activity response results of the target microbial group.

[0013] Optionally, the determination of the optimal culture environment for potential microorganisms within the test object based on microbial taxonomy includes:

[0014] Based on the environment of the object being measured, the types of microorganisms within the object are obtained to obtain information on potential microbial groups;

[0015] Based on information on potential microbial groups, the optimal culture environment for all potential microbial groups is obtained.

[0016] Optionally, multiple groups of optimal culture environments are set up for potential microorganisms, with different proportions of [unspecified ingredient] added to each group of culture environments. 18 A mixture of O-H2O and naturally abundant water was used to obtain an isotopic culture environment for potential microorganisms, including:

[0017] Multiple optimal culture environments for potential microorganisms are configured to obtain backup environments;

[0018] set up 18O isotope gradient, and configuration 18 A mixture of O-H2O and naturally abundant water was used to obtain an isotopic configuration group.

[0019] An isotope configuration group was set up in each backup environment to obtain an isotope culture environment group for potential microorganisms.

[0020] Optionally, the settings 18 O isotope gradient, and configuration 18 A mixture of O-H2O and naturally abundant water was used to obtain isotopic configuration values, including:

[0021] Get 18 Isotopic abundance of O-H2O and naturally abundant water;

[0022] based on 18 The isotopic abundances of O-H2O and naturally occurring water were determined, and the volume ratios were adjusted to adjust the isotopic gradient. The constraint equation for adjusting the isotopic gradient is as follows:

[0023] ,

[0024] Where A represents 18 The volume of O-H2O; B represents the volume of naturally abundant water; V represents the total volume of the mixture. express 18 Isotopic abundance of O-H2O; The isotopic volume of naturally abundant water; Indicates the abundance of the target isotope.

[0025] Optionally, the step of culturing environmental sample microorganisms within the isotopic culture environment group of potential microorganisms to obtain a critical threshold for the response of each potential microorganism includes:

[0026] Microorganisms in the environmental samples to be tested were cultured based on different isotope culture environments.

[0027] Obtain microbial community data under different isotope culture environments;

[0028] Based on the microbial community data, response curves of potential microorganisms and 18O isotope abundance were obtained.

[0029] right 18 The trend of changes in O isotope abundance and microbial response curves was analyzed to identify inflection points or mutation points.

[0030] Obtain all mutation points or inflection points corresponding to each potential microorganism. 18 O isotope abundance was used to obtain the critical threshold for each potential microbial response.

[0031] Optionally, the abundance of stable isotope markers is determined based on the critical threshold of the potential microbial response, and the abundance of each potential microorganism in its optimal culture environment is obtained. 18 O isotope abundance curves include:

[0032] Among the critical thresholds for obtaining microbial responses, the critical threshold that yields the highest results within the same microbial culture time and maintains a stable microbial response after multiple cultures is used as a marker of stable isotope abundance.

[0033] For each potential microorganism, the corresponding stable isotope abundance was introduced into the optimal culture environment, and the abundance at different culture time points was obtained. 18 O isotope abundance;

[0034] Based on the culture time point of potential microorganisms and 18 The correspondence between O isotope abundance was constructed. 18 O isotope abundance curve.

[0035] Optionally, the preprocessing of environmental samples to obtain the sample to be measured includes:

[0036] Environmental samples are acquired and impurities are removed to obtain valid test samples;

[0037] The effective test samples are pretreated by adjusting the moisture conditions, constructing the solution system, and controlling the transfer conditions to obtain the test samples.

[0038] Optionally, the step involves placing the sample to be tested in all optimal culture environments with stable isotope labeling, and obtaining the results under different optimal culture environments. 18 O isotope doping information includes:

[0039] The sample to be tested was separated into liquids, and the number of liquids obtained was the same as the number of all optimal culture environments with stable isotope labels.

[0040] After separation, the samples were placed in all the optimal culture environments with stable isotope labels and cultured.

[0041] Within the same incubation period, obtain all potential microorganisms with stable isotope labels in all optimal culture environments. 18 O isotope doping information.

[0042] Optionally, the target microbial group-based 18 The degree of O isotope incorporation was used to obtain the activity response results of the target microbial group, including:

[0043] Obtain the culture time of the sample to be tested, and obtain all the optimal culture environments corresponding to each culture time. 18 O isotope incorporation information;

[0044] Based on target microbial groups 18 O isotope incorporation information was used to analyze the activity response characteristics of target microbial groups.

[0045] Optionally, the target microbial group-based 18 O isotope incorporation information was used to analyze the activity response results of the target microbial groups, including:

[0046] To obtain the degree of stable isotope incorporation into the target microbial group;

[0047] Obtain the absolute abundance of the target class group in the environmental samples;

[0048] Based on the stable isotope incorporation degree and abundance weight of different target microbial groups, a microbial community growth activity contribution model is constructed, and the total microbial growth activity of environmental samples is estimated based on the model.

[0049] The beneficial effects of this application include:

[0050] 1. Improved effectiveness of the SIP method. In the technical solution of this application, microorganisms are cultured, and different isotopic gradients are set in the culture process. The isotopic incorporation information of the microorganisms obtained in the culture environment with different isotopic gradients and after a period of culture isotope incubation is analyzed. Then, the critical threshold of the microorganism response in different culture processes is analyzed. Based on the obtained threshold, the optimal isotopic configuration parameters can be determined to establish a suitable stable isotopic label abundance.

[0051] 2. The accuracy of the SIP method is guaranteed. In the technical solution of this application, for the stage of determining the abundance of stable isotope markers, based on... 18 In the mixture of O-H2O and naturally abundant water, the volume of these two substances is adjusted to adjust the ratio. At the same time, in determining the isotopic abundance, it is ensured that the isotopic abundance configuration stage of all potential microorganisms is set within the optimal culture environment, thereby ensuring that the only variable in the culture environment is the isotope. Furthermore, the gradient adjustment of isotopic substances is carried out to fully guarantee the accuracy of the determined isotopic labeling abundance.

[0052] 3. The usability of the SIP method is ensured. In the technical solution of this application, the optimal culture environment for each potential microorganism is analyzed and specifically cultured. Furthermore, the isotope label abundance and optimal environment settings are different for different microorganisms. Based on the stable isotope incorporation degree and abundance weight of different microbial groups, a microbial community activity response assessment model is constructed. This enables the assessment of the contribution of different microbial groups to the total growth activity of the microbial community in environmental samples, improving the ability and application of stable isotope probe technology in community activity analysis of complex environmental samples. Attached Figure Description

[0053] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the embodiments of this application or the prior art will be briefly introduced below. Obviously, the following description is only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. The drawings are used to provide a further understanding of this disclosure and constitute a part of the specification. They are used together with the following detailed description to explain this disclosure, but do not constitute a limitation of this disclosure. In the drawings:

[0054] Figure 1 A flowchart illustrating a method for quantifying microbial activity based on stable isotope tracing, provided in this application embodiment;

[0055] Figure 2 A graph illustrating the effect of oxygen isotopes of different isotopic abundances on microbial growth rate in a method for quantifying microbial activity based on stable isotope tracing, provided in this application embodiment.

[0056] Figure 3 A graph illustrating the influence of different isotopic abundances of oxygen isotopes on the structure of microbial functional genes in a method for quantifying microbial activity based on stable isotope tracing, provided in this application embodiment.

[0057] Figure 4 The graph shows the influence of different isotopic abundances of oxygen isotopes on the composition of microbial communities in a method for quantifying microbial activity based on stable isotope tracing provided in this application embodiment. Detailed Implementation

[0058] 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 a part of the embodiments of this application, and not all of them. 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. Furthermore, in the embodiments of this application, "first," "second," etc., are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence.

[0059] In existing environmental sample microbial activity analysis, the common method involves culturing the obtained samples, followed by genetic material detection to analyze the absolute abundance and activity response of microbial communities. While this method yields results, the fixed isotope enrichment conditions used are not suitable for all microbial growth and activity analyses. In some cases, certain microbial communities may exhibit changes in growth activity or metabolic response under high isotope enrichment conditions, leading to biases in the microbial activity analysis results obtained using the SIP method. Furthermore, the SIP method typically does not address the differences in response to isotope enrichment levels among different microbial communities, making it difficult to balance the efficiency of stable isotope labeling with microbial activity perturbation, thus affecting the effectiveness and accuracy of the SIP method.

[0060] To address the problems existing in the prior art, this application discloses a method for quantifying microbial activity based on stable isotope tracing, such as... Figure 1 The diagram shown is a flowchart of a method for quantifying microbial activity based on stable isotope tracing, provided in an embodiment of this application. Specifically:

[0061] S110. Based on the microbial group of the test object, obtain the optimal culture environment for potential microorganisms within the test object.

[0062] S120. Set up multiple groups of optimal culture environments for potential microorganisms, with different proportions of different ingredients added to each group of culture environments. 18 A mixture of O-H2O and naturally abundant water was used to obtain an isotopic culture environment for potential microorganisms.

[0063] S130. The potential microorganisms are cultured in an isotopic culture environment group to obtain the critical threshold of each potential microorganism's response.

[0064] S140. Determine the corresponding stable isotope label abundance based on the critical threshold of the potential microbial response, and obtain the abundance of each potential microorganism in its optimal culture environment. 18 O isotope abundance curve.

[0065] S150. Pre-process the environmental samples to obtain the samples to be measured.

[0066] S160. Place the sample to be tested in all optimal culture environments with stable isotope labeling, and obtain the results under different optimal culture environments. 18 O isotope doping information.

[0067] S170, Based on target microbial groups 18 The degree of O isotope incorporation was used to obtain the activity response results of the target microbial group.

[0068] The beneficial effects of the above steps are that they can ensure the effectiveness and accuracy of the SIP method. At the same time, after determining the stable isotope label abundance of each potential microorganism, the stable isotope label abundance can be optimized based on the response threshold, and the stable isotope incorporation information of the target microbial group can be obtained based on the optimized label abundance, thereby improving the reliability of microbial activity analysis and community activity assessment.

[0069] The following will provide a detailed explanation and description of all the steps above:

[0070] As described in step S110, the purpose of this step is to determine the types of microorganisms present in various tested objects based on existing research results. This allows for the determination of the optimal culture environment for each type of microorganism based on the information on potential microbial species, thus establishing the foundation for subsequent culture environment configuration. Specifically:

[0071] S111. Based on the environment of the object being measured, obtain the types of microorganisms within the object being measured in order to obtain information on potential microbial groups.

[0072] The purpose of this step is to identify the various microbial groups that may exist in the environment being tested, based on which the optimal environment can be constructed.

[0073] Specifically, for different tested environments, the types of microorganisms in various environments, as described in existing research results, are obtained.

[0074] This requires establishing corresponding microbial species information for each type of environment, such as the correspondence between water and microorganisms, the correspondence between air and microorganisms, and the correspondence between soil and microorganisms.

[0075] In some embodiments, all currently identified common microorganisms can be identified as potential microorganisms in the environment. However, this method requires higher data processing capabilities and culture system support capabilities for subsequent detection systems, which is often difficult to support technically and needs to be selected according to the actual performance of the laboratory.

[0076] S112. Based on the information of potential microbial groups, obtain the optimal culture environment for all potential microbial groups.

[0077] The purpose of this step is to obtain potential microbial groups and thus potential microbial species. The optimal culture environment for each type of microorganism has already been determined in existing studies, so it is necessary to determine the optimal culture environment.

[0078] Among them, considering that the isotope probe in this application is based on 18 O-H2O is obtained from naturally abundant water, so the water content of the culture material or the amount of water added should also be taken as an optimal culture environment parameter.

[0079] In some embodiments, the optimal culture environment obtained can also be eliminated. 18 O-H2O and natural abundance water factors, that is, eliminating water resources in the culture environment and using all other factors as culture environment factors, and selecting the best culture parameters from them.

[0080] The beneficial effect of step S110 is that it eliminates the need for black-box verification of the environment being tested, and instead allows for the direct use of existing research results to set the optimal culture environment, thereby laying the foundation for subsequent work on isotope probe determination and culture environment determination.

[0081] As described in step S120, the purpose of this step is to configure the isotopic abundance and set up the culture environment group for potential microorganisms, thereby culturing the potential microorganisms after determining the corresponding culture environment group. Specifically:

[0082] S121. Configure multiple sets of optimal culture environments for potential microorganisms to obtain backup environments.

[0083] The purpose of this step is to obtain more accurate analytical results, which requires... 18 A mixture of O-H2O and naturally abundant water was prepared and used. The abundance of isotope labeling was then determined. To validate this, it is clearly necessary to establish optimal culture environments for multiple potential microorganisms and conduct [further testing / processing]. 18 The addition of O-H2O to a mixture of naturally abundant water.

[0084] For each potential microorganism, after determining its optimal culture environment, multiple sets of optimal culture environments need to be configured.

[0085] Among them, when the optimal culture environment is obtained, it does not contain 18 When using a mixture of O-H2O and naturally abundant water, the optimal culture environment should be maintained using the same configuration. However, if other components are present, it is necessary to ensure... 18 The O-H2O mixture differs from the mixture of naturally abundant water, while all other culture environment parameters are exactly the same.

[0086] This requires configuring multiple sets of optimal culture environments for each potential microorganism to obtain all available backup environments. These culture environments include all factors that affect microbial growth, such as culture medium, ambient temperature, ambient humidity, and light intensity.

[0087] S122, Settings 18 O isotope gradient, and configuration 18 A mixture of O-H2O and naturally abundant water was used to obtain an isotopic configuration.

[0088] The purpose of this step is to... 18 A mixture of O-H2O and naturally abundant water was prepared, and based on the obtained preparation gradient, subsequent stable isotope probe identification was performed within the obtained preparation gradient. Specifically:

[0089] S1221, Obtain 18 Isotopic abundance of O-H2O and naturally abundant water.

[0090] The purpose of this step is to, for 18 The specific values ​​of O-H2O and naturally abundant water are determined so that the radioactivity abundance can be determined in subsequent analyses based on volume adjustments.

[0091] Among them, can 18 The isotopic abundance of O isotope-labeled water is 97.8 atom, while the isotopic abundance of naturally abundant ultrapure water is 0.2 atom.

[0092] S1222, based on 18 The isotopic abundances of O-H2O and naturally occurring water were determined, and the volume ratios were adjusted to adjust the isotopic gradient. The constraint equation for adjusting the isotopic gradient is as follows:

[0093] ,

[0094] Where A represents 18 The volume of O-H2O; B represents the volume of naturally abundant water; V represents the total volume of the mixture. express18 Isotopic abundance of O-H2O; The isotopic volume of naturally abundant water; Indicates the abundance of the target isotope.

[0095] The purpose of this step is to obtain... 18 The isotopic abundances of O-H2O and naturally abundant water were adjusted, and after determining the abundance values ​​of the two isotopes, the value was determined, thereby performing gradient adjustment based on the set constraints.

[0096] Within the set of equations, it is necessary to ensure that the total volume is the same, and 18 The volume of O-H2O and naturally abundant water is adjusted to adjust the radioactivity abundance.

[0097] The abundance gradient adjustment can be set to 0.2 atom%, 5 atom%, 10 atom%, 20 atom%, 30 atom%, 40 atom%, 50 atom%, 60 atom%, 70 atom%, and 80 atom%.

[0098] Natural abundance-treated samples and isotope-labeled samples should be handled and cultured separately to avoid cross-contamination of isotopes.

[0099] Taking a soil sample as an example, the soil sample was divided into ten groups, with each group containing 2 g of fresh soil. In the gradient setting, soil samples reaching a preset moisture content range were divided into multiple groups, and soil samples from each group were weighed and placed into culture bottles. Additives were then added to each group of soil samples. 18 A mixture of O-H2O isotope-labeled water and naturally abundant ultrapure water was used to create soil samples with varying isotope abundances by adjusting the volume ratio of the two solutions. 18 O isotope gradient; the 18 The O isotope enrichment gradient included multiple different levels; then, each group of soil samples was placed in a constant temperature incubator for cultivation. After adding a mixture of isotope-labeled water and naturally abundant water, the soil moisture content was brought to 60% of field capacity. The cultivation temperature was 15℃ (or the temperature required according to the experimental design). During the cultivation period, the soil moisture was kept constant to ensure the stability of the microbial metabolic process.

[0100] In one specific example, the environmental sample is a soil sample, and the acquisition process involves different methods. 18 The soil samples labeled with O isotope gradients included: dividing soil samples that reached a preset range of moisture content into multiple groups, weighing soil samples from each group and placing them into culture bottles; adding O isotope gradient labeling to each group of soil samples. 18A mixture of O-H2O isotope-labeled water and naturally abundant ultrapure water was used to create soil samples with varying isotope abundances by adjusting the volume ratio of the two solutions. 18 O isotope gradient; the 18 The O isotope enrichment gradient included multiple different levels; subsequently, each group of soil samples was placed in a constant temperature incubator for incubation.

[0101] It should be noted that the above 18 The isotopic abundance of O isotope-labeled water is 97.8 atom, while the isotopic abundance of naturally abundant ultrapure water is 0.2 atom.

[0102] It should be noted that the soil samples were divided into ten groups, and each treatment sample in each group was weighed out as 2 g of fresh soil.

[0103] It should be noted that the gradient includes: 0.2 atom%, 5 atom%, 10 atom%, 20 atom%, 30 atom%, 40 atom%, 50 atom%, 60 atom%, 70 atom%, and 80 atom%.

[0104] It should be noted that the addition of a mixture of isotope-labeled water and naturally abundant water brought the soil moisture content to 60% of field capacity.

[0105] It should be noted that the incubation temperature is 15℃ (or the temperature required by the experimental design), and the soil moisture should be kept constant during the incubation period to ensure the stability of the microbial metabolic process;

[0106] It should be noted that samples treated with natural abundance and samples treated with isotope labeling should be handled and cultured separately to avoid cross-contamination of isotopes.

[0107] S123. Set up isotope configuration groups in each backup environment to obtain isotope culture environment groups for potential microorganisms.

[0108] The purpose of this step is to construct the environmental genome after obtaining the isotopic configuration, so that potential microbial cultures can be cultured in subsequent analyses.

[0109] The potential microorganisms obtained are cultured in an optimal culture environment, and the only difference between each culture environment is the isotope abundance value.

[0110] This requires distributing the product according to the isotopic abundance gradient within the configured optimal environment. 18 A mixture of O-H2O and naturally abundant water.

[0111] The beneficial effect of step S120 is that it allows for the determination of isotope probes by adjusting the isotope abundance gradient and then configuring the optimal culture environment for potential microorganisms.

[0112] As described in step S130, the purpose of this step is to determine the response threshold of all potential microorganisms during the culture of each potential microorganism, from which the abundance of stable isotope markers can be selected. Specifically:

[0113] S131. Based on different isotope culture environment groups, microorganisms in the environmental samples to be tested are cultured.

[0114] The purpose of this step is to allow for the cultivation of microorganisms within the isotope culture environment after the environment has been configured, so that nucleic acid extraction can be performed after effective culture operations to determine the microbial sequence information.

[0115] This requires the construction of the environment based on the optimal culture environment information for each potential microorganism.

[0116] This requires, after constructing the optimal culture environment, the following steps: 18 A mixture of O-H2O and naturally abundant water was used to set up isotope labeling gradients with different ratios.

[0117] In this process, after setting up isotope labeling gradients with different ratios, microorganisms in the environmental samples to be tested were cultured.

[0118] S132. Obtain the microbial response data of the isotope culture environment group corresponding to each potential microorganism, and obtain... 18 O isotope abundance and microbial response curves.

[0119] The purpose of this step is to determine the microbial response data of each potential microorganism and plot it as a curve, thereby identifying the points of change in the growth and activity information of potential microorganisms, especially to obtain the critical threshold, which can then be used to obtain the abundance of stable isotope labeling.

[0120] This requires detecting the isotopic abundance of potential microorganisms at specific time points during the analysis process to obtain the results.

[0121] This requires combining the obtained test results with the corresponding... 18 The parameters of the mixture of O-H2O and naturally abundant water were determined to obtain... 18 Correlation between O isotope abundance and microbial response.

[0122] In the measurement of isotope abundance, nucleic acid detection is used. The steps include: culturing soil samples labeled with different isotope enrichment gradients; extracting and processing nucleic acids from the cultured soil samples; and detecting the isotope abundance of the nucleic acid samples to obtain different... 18 The degree of isotopic labeling of nucleic acids under O isotope abundance.

[0123] In nucleic acid testing, nucleic acid extraction requires purification and drying to remove moisture and impurities that could affect the accuracy of isotope detection.

[0124] It should be noted that the abundance of the nucleic acid isotopes can be determined by isotope ratio mass spectrometry (IRMS).

[0125] It should be noted that the analysis of the microbial community composition can be achieved through high-throughput sequencing technology, including but not limited to 16S rRNA gene sequencing or ITS sequencing.

[0126] It should be noted that the microbial functional structure response can be obtained through functional gene or functional annotation analysis, including functional genes related to carbon cycling, nitrogen cycling, etc.

[0127] It should be noted that the community structure and functional response data are used to reveal the changing characteristics of microbial composition and potential functions under different 18O isotope abundances.

[0128] Among the measurements of soil samples, the metabolic activity of the soil samples may be measured, including the respiration rate.

[0129] Among them, it is necessary to obtain the 18 The correspondence between O isotope abundance and microbial response level is constructed in a coordinate system to generate... 18 O isotope abundance and microbial response curves.

[0130] S133, to 18 The changes in O isotope abundance and microbial response curves were analyzed to identify inflection points or mutation points.

[0131] The purpose of this step is to consider, in determining stable isotope probes, both the microbial proliferation process and its upper limit for isotope uptake; upon reaching these states, [they will...]. 18 The O isotope abundance and microbial response curves show inflection points or mutation points, so it is necessary to obtain subsequent key information based on the determination of inflection points and mutation points.

[0132] Among them, it is necessary to obtain the 18The trends in O isotope abundance and microbial response curves were analyzed to determine the underlying causes. 18 Trends in O isotope abundance.

[0133] This requires, based on the observed trends, to... 18 The abundance of O isotopes and the inflection points and / or mutation points in the microbial response curves are determined to assess the microbial growth status.

[0134] S134. Obtain all mutation points or inflection points corresponding to each potential microorganism. 18 O isotope abundance was used to obtain the critical threshold for each potential microbial response.

[0135] The purpose of this step is to determine the critical threshold for potential microbial responses, which can then be used to determine the abundance of stable isotope labeling.

[0136] Among them, the corresponding information for each potential microorganism was obtained. 18 All mutation points and / or inflection points in the O isotope abundance and microbial response curves, and obtain the corresponding mutation points and / or inflection points. 18 O isotope abundance.

[0137] Among them, the mutation points and / or inflection points corresponding to the obtained mutation points and / or inflection points 18 The abundance of O isotopes is directly set as the critical threshold for the potential microbial response.

[0138] In the process of acquiring information on microbial growth, the completion of... 18 Environmental samples labeled with O isotope gradients were cultured or incubated to obtain different... 18 Microbial activity data under O isotope abundance, including microbial growth rate, microbial community composition, and response data of community functional structure.

[0139] In a specific example, the environmental sample is a soil sample, and the acquisition of microbial activity data includes: culturing soil samples labeled with different isotope enrichment gradients, extracting and processing nucleic acids from the cultured soil samples, and detecting the isotope abundance of the nucleic acid samples to obtain different... 18 The degree of isotopic labeling of nucleic acids under O isotope abundance;

[0140] Based on the nucleic acid 18 The degree of O incorporation is used to calculate the microbial growth rate;

[0141] Soil samples were analyzed for community structure and function to obtain characteristics of microbial communities and their functional responses.

[0142] It should be noted that nucleic acid extraction requires purification and drying to remove moisture and impurities that could affect the accuracy of isotope detection.

[0143] It should be noted that the abundance of the nucleic acid isotopes can be determined by isotope ratio mass spectrometry (IRMS).

[0144] It should be noted that the analysis of the microbial community composition can be achieved through high-throughput sequencing technology, including but not limited to 16S rRNA gene sequencing or ITS sequencing.

[0145] It should be noted that the microbial functional structure response can be obtained through functional gene or functional annotation analysis, including functional genes related to carbon cycling, nitrogen cycling, etc.

[0146] It should be noted that community structure and functional response data are used to reveal different 18 Characteristics of changes in microbial composition and potential functions under O isotope abundance;

[0147] It should be noted that the soil samples may be subjected to metabolic activity measurements, including respiration rate measurements.

[0148] The beneficial effect of step S130 is that, through the... 18 The determination of O isotope abundance and microbial response curves allows for the identification of a critical threshold for each potential microbial response, which can then be used to determine the subsequent stable isotope labeling abundance.

[0149] As described in step S140, the purpose of this step is that, after obtaining the stable isotope label abundance in steps S110-S130, it is necessary to conduct different incubation times based on this abundance. 18 O isotope abundance curves were used to establish the data foundation for subsequent microbial taxonomy determination. Specifically:

[0150] S141. Among the critical thresholds for obtaining microbial responses, the critical threshold that yields the highest results within the same microbial culture time and maintains a stable microbial response after multiple cultures is used as the stable isotope label abundance.

[0151] The purpose of this step is that, after the critical threshold of the response has been determined, the corresponding isotopic abundance markers need to be set based on the results obtained, so that the set isotopic abundances can be used to lay the foundation for subsequent isotopic abundance curve determination.

[0152] Among them, for each potential microorganism, the critical threshold of the microbial response obtained from multiple culture results in the same culture environment during the culture process.

[0153] Among the multiple thresholds obtained, it is necessary to compare the generated thresholds and select the one that consistently appears and has the highest threshold value. 18 A mixed solution addition scheme for O-H2O and naturally abundant water.

[0154] Among them, it is necessary to obtain 18 After the mixture of O-H2O and naturally abundant water was added, the mixture was used as an isotope labeling abundance.

[0155] The response relationship construction and threshold identification methods are as follows: based on different 18 Microbial activity data obtained at O ​​isotope abundance were used to construct the relationship between isotope abundance and microbial response, and inflection points or mutation points that significantly changed the relationship were identified. 18 The abundance of O isotopes was determined as the critical threshold.

[0156] In a specific example, the response relationship construction and threshold identification include: based on different 18 Microbial response data obtained under O isotope abundance were used to construct... 18 The relationship between O isotope abundance and microbial response;

[0157] Trend analysis was performed on the aforementioned relationship to identify the changes in microbial response. 18 The turning point or mutation point in the process of significant changes in O isotope abundance;

[0158] The turning point or mutation point corresponding to 18 O isotope abundance was determined as a critical threshold for microbial response;

[0159] When multiple inflection points exist, one or more critical thresholds are determined to characterize the microbial response. 18 Different response stages of O isotope enrichment.

[0160] It should be noted that the trend analysis can be achieved through nonlinear fitting, piecewise regression models, or change point analysis methods.

[0161] It should be noted that the microbial activity data includes one or more of the following: microbial growth rate, microbial community composition, and microbial functional gene data.

[0162] S142. For each potential microorganism, apply the corresponding stable isotope label abundance within the optimal culture environment, and obtain the corresponding abundance at different culture time points. 18 O isotope doping information.

[0163] The purpose of this step is that, after the isotope probes are determined, the corresponding isotope abundance label needs to be used for each potential microorganism to determine the isotope abundance at different culture time points of the potential microorganism, so as to show the change of isotope abundance of different potential microorganisms over time.

[0164] In the cultivation process of each potential microorganism, the corresponding isotope abundance is introduced into the optimal culture environment for each potential microorganism.

[0165] In the cultivation process of each potential microorganism, it is necessary to monitor the isotope abundance obtained at different cultivation times.

[0166] This requires establishing the culture time for potential microorganisms. 18 The correspondence between O isotope abundance, etc. Figure 2 The figure shown is a graph illustrating the effect of different isotopic abundances of oxygen isotopes on microbial growth rate in a method for quantifying microbial activity based on stable isotope tracing provided in this application embodiment.

[0167] The isotope label abundance is determined by: determining the SIP method based on the critical threshold. 18 The reasonable isotopic abundance of O isotope labeling is determined, and isotope labeling is performed according to the said isotopic abundance.

[0168] In a specific example, the determination of the isotope label abundance includes: selecting elements below the critical threshold based on one or more critical thresholds obtained in step four. 18 The abundance of O isotope is used as the isotope labeling abundance, and isotope labeling is performed according to the said isotope abundance.

[0169] It should be noted that the abundance of the isotope labeling is preferably below the critical threshold. 18 O isotope abundance.

[0170] S143, Based on the culture time point of potential microorganisms and 18 The correspondence between O isotope abundance was constructed. 18 O isotope abundance curve.

[0171] The purpose of this step is to... 18 The O isotope abundance curve is then plotted, allowing for the execution of specific measurement and analysis based on this curve information in subsequent group detection.

[0172] Among them, the culture time points of potential microorganisms were obtained and 18 After determining the correspondence between O isotope abundances, these two values ​​need to be set within the coordinate system.

[0173] In this regard, considering that it is impossible to perform non-killing isotopic abundance detection on potential microorganisms in the determination of potential microorganisms, but rather it is necessary to monitor them by killing them and detecting their genetic material, therefore the culture time points of the potential microorganisms and 18 The abundance correspondence of O isotopes is often represented by discrete points. In such cases, interpolation can be used within a coordinate system to construct the entire curve. For example... Figure 3 The figure shown is a graph illustrating the influence of different isotopic abundances of oxygen isotopes on the structure of microbial functional genes in a method for quantifying microbial activity based on stable isotope tracing provided in an embodiment of this application.

[0174] This requires constructing isotope abundance curves for each potential microorganism, which can then be used for subsequent microbial group analysis.

[0175] In some embodiments, stable isotope-labeled cultures can be directly performed on mixed microbial communities or environmental samples from different ecosystems, and isotope incorporation information can be measured to ultimately establish a correlation between stable isotope enrichment levels and microbial community responses. For complex environmental samples, this method can directly reflect the overall community response changes under different isotope enrichment conditions, thereby determining the impact of isotope enrichment levels on community structure, activity responses, or functional responses. Figure 4 The figure shows the influence curves of different isotopic abundances of oxygen isotopes on microbial community composition in a method for quantifying microbial activity based on stable isotope tracing provided in an embodiment of this application. However, relatively speaking, when analyzing directly based on the overall community response, the results obtained may be affected by the initial community composition of the sample, differences in responses among different taxa, and differences in culture conditions. In subsequent practical applications, if the community composition of the environmental sample to be tested differs significantly from the sample used when establishing the response relationship, the comparability of the community response results may be reduced. Therefore, when using this method, it is preferable to combine the isotopic incorporation information, abundance weights, and response thresholds of the target microbial taxa for comprehensive analysis to improve the accuracy and applicability of the microbial community activity assessment results.

[0176] The beneficial effect of step S140 is that by determining the isotope abundance curve, the enrichment of isotope abundance of different types of potential microorganisms can be directly determined, and the composition of microorganisms can be determined directly based on the obtained measurement curve in subsequent microbial group determination.

[0177] As described in step S150, the purpose of this step is to pre-process the obtained sample before performing the actual detection operation to ensure that the sample can be used in the actual measurement process. Specifically:

[0178] S151. Obtain environmental samples and remove impurities to obtain valid test samples.

[0179] The purpose of this step is to remove impurities from the obtained sample to avoid the impurities causing a decrease in the accuracy of the measurement results.

[0180] Different impurity removal methods need to be used depending on the type of environmental sample, such as soil, air, and water. It is important to note that the methods used should not cause the death of microorganisms.

[0181] After impurity removal, the samples being tested need to be protected to prevent the death of microorganisms.

[0182] After determining the sample type, the potential microorganisms within it are identified, and the corresponding isotopic abundance curves of the potential microorganisms are retrieved.

[0183] S152. Adjust the moisture conditions, construct the solution system, and regulate the transfer conditions of the effective test samples to pre-treat the effective test samples and obtain the samples to be tested.

[0184] The purpose of this step is to further preprocess the sample after impurity removal so that the obtained sample can be used in subsequent actual culture to obtain sufficient test results.

[0185] This requires determining the types and quantities of potential microorganisms present in the sample based on the type of sample being tested.

[0186] After determining the number of potential microbial species, the obtained valid test samples are divided into n equal parts, which is the same as the number of potential microbial species.

[0187] Among all the pretreatment operations, the content includes moisture condition adjustment, solution system construction and transfer condition control.

[0188] In some embodiments, the valid test samples may be proliferated before pretreatment to ensure that the allocated samples contain a sufficient amount of the test microorganisms.

[0189] The beneficial effect of step S150 is that the samples can be cultured separately by pre-processing and distributing the samples to be measured, thereby ensuring that the accuracy of the test results is not affected by the presence of impurities.

[0190] As described in step S160, the purpose of this step is to culture the obtained samples to be tested separately and independently, and then obtain the isotope abundance at different culture times, thereby determining the relationship between the total isotope incorporation level and the abundance curves of each potential microorganism in different environments, for the purpose of measurement. Specifically:

[0191] S161. The sample to be tested is separated into liquids, and the number of liquids obtained is the same as the number of all optimal culture environments with stable isotope probes.

[0192] The purpose of this step is to culture the samples in all the constructed culture environments for the samples to be tested, and to ensure that each constructed environment contains one unit of sample.

[0193] After determining the optimal culture environment and isotope labeling abundance for each type of potential microorganism, it is necessary to set the corresponding isotope abundance within the optimal culture environment.

[0194] This requires setting up the sample to be tested in each environment according to the optimal culture environment corresponding to all potential microorganisms, after liquid separation.

[0195] In particular, for the culture environment of the sample to be tested, it is necessary to establish a correspondence between these two types of information in order to conduct subsequent measurements.

[0196] S162. Place the separated samples into all the optimal culture environments with stable isotope labels and culture them.

[0197] The purpose of this step is to actually culture the obtained sample to obtain the microbial taxa. 18 O isotope incorporation into nucleic acids.

[0198] This process requires culturing the samples to be tested for a period of time before proceeding. 18 Acquisition of information on O isotope doping.

[0199] S163. Within the same incubation period, obtain the microorganisms in all optimal incubation environments with stable isotope labels. 18 O isotope doping information.

[0200] The purpose of this step is to... 18 By obtaining information on the incorporation of O isotopes, the growth and activity of microbial groups can then be analyzed based on the measured values.

[0201] In this regard, it is necessary to ensure that the culture time of the environmental samples under different stable isotope enrichment gradients is consistent, so as to reduce the impact of the difference in culture time on the stable isotope incorporation results and obtain the stable isotope incorporation information of microorganisms under different culture conditions.

[0202] In order to improve the accuracy of stable isotope incorporation information analysis, it is necessary to determine the stable isotope enrichment threshold at which the microbial response changes significantly based on the response data of microorganisms under different stable isotope enrichment conditions, and select an appropriate stable isotope labeling abundance range based on the threshold.

[0203] After reaching the required culture time, it is necessary to obtain the degree of stable isotope incorporation of microbial nucleic acid molecules under different stable isotope enrichment conditions, and analyze the growth and activity response characteristics of the target microbial group based on the degree of stable isotope incorporation.

[0204] The beneficial effect of step S160 is that by acquiring and analyzing information on the stable isotope incorporation of microorganisms in all optimal culture environments, a microbial community activity response analysis model can be established based on the stable isotope response characteristics of different microbial groups, thereby improving the accuracy and efficiency of microbial community activity analysis and reducing the impact of high isotope enrichment conditions on microbial activity response during stable isotope labeling.

[0205] As described in step S170, the purpose of this step is to analyze the activity response results of the target microbial group based on the degree of stable isotope incorporation, in order to obtain the characteristics of microbial activity response. Specifically:

[0206] S171. Obtain the degree of stable isotope incorporation of the target microbial group.

[0207] The purpose of this step is to detect stable isotope incorporation information in the target microbial community in the environmental sample to be tested, so as to obtain the degree of stable isotope incorporation of the target microbial community under different stable isotope enrichment conditions.

[0208] After determining the culture time of the sample to be tested, it is necessary to perform stable isotope incorporation detection on all target microbial groups in the culture environment to reduce the impact of culture time differences on the test results.

[0209] This requires ensuring that the samples to be tested within all optimal culture environments are within the range of... 18 Simultaneous measurement of the O isotope nucleation doping ensures data comparability.

[0210] In this process, it is also necessary to determine the obtained 18O incorporation information, the corresponding culture environment information, and the various potential microorganisms corresponding to that culture environment information. 18 O isotope abundance curve.

[0211] S172. Obtain the culture time points of the samples to be measured, in all... 18 The corresponding O isotope abundance curve 18 O-incorporation of nucleic acids to obtain theoretical microorganisms 18 O assimilates data.

[0212] The purpose of this step is to... 18 The abundance of O isotopes is selected, and then the composition of the sample to be measured can be established. 18 The information incorporated into the nucleic acid corresponds to the growth and other activity data obtained from each optimal culture environment. Only after obtaining this value can subsequent calculations of microbial activity groups be performed.

[0213] S173. Based on the degree of stable isotope incorporation into the target microbial group, obtain the activity response results of the target microbial group.

[0214] The purpose of this step is to evaluate the bioactivity of the target microbial community based on the degree of stable isotope incorporation and its response characteristics.

[0215] Among them, the contribution of the target microbial group to the total growth activity of the microbial community can be analyzed by combining the degree of stable isotope incorporation of the target microbial group and the abundance information of the target microbial group.

[0216] Specifically, the activity response pattern of microbial communities can be evaluated based on the degree of stable isotope incorporation and abundance weight of different target microbial groups. The beneficial effect of step S170 is that it directly uses the obtained stable isotope label abundance to determine the information of microbial groups contained in the sample being measured, without the need for independent genetic material detection, thereby significantly improving the efficiency of microbial group measurement and ensuring measurement accuracy and validity.

[0217] The beneficial effects of this application include:

[0218] 1. Improved effectiveness of the SIP method. In the technical solution of this application, microorganisms are cultured, and different isotopic gradients are set in the culture process. The isotopic incorporation information of the microorganisms obtained in the culture environment with different isotopic gradients and after a period of culture isotope incubation is analyzed. Then, the critical threshold of the microorganism response in different culture processes is analyzed. Based on the obtained threshold, the optimal isotopic configuration parameters can be determined to establish a suitable stable isotopic label abundance.

[0219] 2. The accuracy of the SIP method is guaranteed. In the technical solution of this application, for the stage of determining the abundance of stable isotope markers, based on... 18 In the mixture of O-H2O and naturally abundant water, the volume of these two substances is adjusted to adjust the ratio. At the same time, in determining the isotopic abundance, it is ensured that the isotopic abundance configuration stage of all potential microorganisms is set within the optimal culture environment, thereby ensuring that the only variable in the culture environment is the isotope. Furthermore, the gradient adjustment of isotopic substances is carried out to fully guarantee the accuracy of the determined isotopic labeling abundance.

[0220] 3. The usability of the SIP method is ensured. In the technical solution of this application, the optimal culture environment for each potential microorganism is analyzed and specifically cultured. Furthermore, the isotope label abundance and optimal environment settings are different for different microorganisms. Based on the stable isotope incorporation degree and abundance weight of different microbial groups, a microbial community activity response assessment model is constructed. This enables the assessment of the contribution of different microbial groups to the total growth activity of the microbial community in environmental samples, improving the ability and application of stable isotope probe technology in community activity analysis of complex environmental samples.

[0221] Those skilled in the art will understand that all or part of the steps of the above method embodiments can be implemented by hardware related to computer program instructions. The aforementioned computer program can be stored in a non-volatile storage medium, and when executed, it performs the steps of the above method embodiments. Alternatively, if the integrated unit of the present invention is implemented as a software functional module and sold or used as an independent product, it can also be stored in a non-volatile storage medium. Based on this understanding, the technical solution of the embodiments of the present invention, or the part that contributes to the prior art, can be embodied in the form of a software product. This computer software product is stored in a non-volatile storage medium and includes several instructions to cause an electronic device (which may be a personal computer, server, network device, etc.) to execute all or part of the methods described in the various embodiments of the present invention.

[0222] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for quantifying microbial activity based on stable isotope tracing, characterized in that, The determination method includes: Based on the microbial group of the test object, obtain the optimal culture environment for potential microorganisms within the test object; Multiple optimal culture environments were set up for potential microorganisms, with different proportions of [unspecified ingredient] added to each culture environment. 18 A mixture of O-H2O and naturally abundant water was used to obtain an isotopic culture environment for potential microorganisms. Environmental microorganisms were cultured within an isotopic culture environment group of potential microorganisms to obtain the critical threshold of response for each potential microorganism. The abundance of stable isotope markers was determined based on the critical threshold of potential microbial responses, and the abundance of each potential microorganism in its optimal culture environment was obtained. 18 O isotope abundance curve; Environmental samples are pretreated to obtain the samples to be tested; The samples to be tested were placed in all the optimal culture environments with stable isotope labeling, and the results were obtained in different optimal culture environments. 18 O isotope incorporation information; Based on target microbial groups 18 The degree of O isotope incorporation was used to obtain the activity response results of the target microbial group.

2. The method for quantifying microbial activity based on stable isotope tracing according to claim 1, characterized in that, The determination of the target microbial group, including obtaining the optimal culture environment for potential microorganisms within the target microorganism, comprises: Based on the environment of the object being measured, the types of microorganisms within the object are obtained to obtain information on potential microbial groups; Based on information on potential microbial groups, the optimal culture environment for all potential microbial groups is obtained.

3. The method for quantifying microbial activity based on stable isotope tracing according to claim 1, characterized in that, Multiple optimal culture environments were set up for the potential microorganisms, with different proportions of different ingredients added to each culture environment. 18 A mixture of O-H2O and naturally abundant water was used to obtain an isotopic culture environment for potential microorganisms, including: Multiple optimal culture environments for potential microorganisms are configured to obtain backup environments; set up 18 O isotope gradient, and configuration 18 A mixture of O-H2O and naturally abundant water was used to obtain an isotopic configuration group. An isotope configuration group was set up in each backup environment to obtain an isotope culture environment group for potential microorganisms.

4. The method for quantifying microbial activity based on stable isotope tracing according to claim 3, characterized in that, The settings 18 O isotope gradient, and configuration 18 A mixture of O-H2O and naturally abundant water was used to obtain isotopic configuration values, including: Get 18 Isotopic abundance of O-H2O and naturally abundant water; based on 18 The isotopic abundances of O-H2O and naturally occurring water were determined, and the volume ratios were adjusted to adjust the isotopic gradient. The constraint equation for adjusting the isotopic gradient is as follows: ， Where A represents 18 The volume of O-H2O; B represents the volume of naturally abundant water; V represents the total volume of the mixture. express 18 Isotopic abundance of O-H2O; The isotopic volume of naturally abundant water; Indicates the abundance of the target isotope.

5. The method for quantifying microbial activity based on stable isotope tracing according to claim 1, characterized in that, The step of culturing environmental microorganisms within an isotopic culture environment group of potential microorganisms to obtain a critical threshold for the response of each potential microorganism includes: Microorganisms in the environmental samples to be tested were cultured based on different isotope culture environments. Obtain microbial community data under different isotope culture environments; Based on the aforementioned whole microbial community data, potential microorganisms and 18 Response curve of O isotope abundance; right 18 The trend of changes in O isotope abundance and microbial response curves was analyzed to identify inflection points or mutation points. Obtain all mutation points or inflection points corresponding to each potential microorganism. 18 O isotope abundance was used to obtain the critical threshold for each potential microbial response.

6. The method for quantifying microbial activity based on stable isotope tracing according to claim 1, characterized in that, The critical threshold based on the potential microbial response is used to determine the corresponding stable isotope label abundance, and the abundance of each potential microorganism in its optimal culture environment is obtained. 18 O isotope abundance curves include: Among the critical thresholds for obtaining microbial responses, the critical threshold that yields the highest results within the same microbial culture time and maintains a stable microbial response after multiple cultures is used as a marker of stable isotope abundance. For each potential microorganism, the corresponding stable isotope abundance was introduced into the optimal culture environment, and the abundance at different culture time points was obtained. 18 O isotope abundance; Based on the culture time point of potential microorganisms and 18 The correspondence between O isotope abundance was constructed. 18 O isotope abundance curve.

7. The method for quantifying microbial activity based on stable isotope tracing according to claim 1, characterized in that, The preprocessing of environmental samples to obtain the sample to be measured includes: Environmental samples are acquired and impurities are removed to obtain valid test samples; The effective test samples are pretreated by adjusting the moisture conditions, constructing the solution system, and controlling the transfer conditions to obtain the test samples.

8. The method for quantifying microbial activity based on stable isotope tracing according to claim 1, characterized in that, The process involves placing the sample to be tested in all optimal culture environments with stable isotope labeling, and obtaining data from different optimal culture environments. 18 O isotope doping information includes: The sample to be tested was separated into liquids, and the number of liquids obtained was the same as the number of all optimal culture environments with stable isotope labels. After separation, the samples were placed in all the optimal culture environments with stable isotope labels and cultured. Within the same incubation period, obtain all potential microorganisms with stable isotope labels in all optimal culture environments. 18 O isotope doping information.

9. The method for quantifying microbial activity based on stable isotope tracing according to claim 1, characterized in that, The target microbial group-based 18 The degree of O isotope incorporation was used to obtain the activity response results of the target microbial group, including: Obtain the culture time of the sample to be tested, and obtain all the optimal culture environments corresponding to each culture time. 18 O isotope incorporation information; Based on target microbial groups 18 O isotope incorporation information was used to analyze the activity response characteristics of target microbial groups.

10. The method for quantifying microbial activity based on stable isotope tracing according to claim 9, characterized in that, The target microbial group-based 18 O isotope incorporation information was used to analyze the activity response results of the target microbial group, including obtaining the degree of stable isotope incorporation of the target microbial group. Obtain the absolute abundance of the target class group in the environmental samples; Based on the stable isotope incorporation degree and abundance weight of different target microbial groups, a microbial community growth activity contribution model is constructed, and the total microbial growth activity of environmental samples is estimated based on the model.

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