Analysis method for carbon synthesis rate and carbon utilization efficiency of extracellular polymeric substance

Abstract

The invention provides a method for analyzing the carbon synthesis rate and the carbon utilization efficiency of an extracellular polymeric substance, and relates to the technical field of analytical testing, the method for analyzing the carbon synthesis rate and the carbon utilization efficiency of the extracellular polymeric substance comprises the following steps: pre-culturing sampled soil; adding isotope < 18 > O-H2O with the purity of 98.0 at% to enable the content of < 18 > O-H2O in the soil to be 20 at% as a marking group; meanwhile, setting a control group in which the isotope 18O-H2O is not added; extracting extracellular polymeric substances of the soil of the marking group and the control group by using a cation exchange resin method, and separating and purifying to obtain purified polysaccharide and protein; measuring the abundance and total oxygen content of 18O in the purified polysaccharide and protein samples by using an isotope mass spectrometer, and respectively calculating the atomic percentage excess of the purified polysaccharide and protein; calculating the yield of the purified polysaccharide and protein; and calculating the carbon synthesis rate and the carbon utilization efficiency of the extracellular polymeric substance.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and testing, and in particular to a method for analyzing extracellular polymer carbon synthesis rate and carbon utilization efficiency. Background Art

[0002] Extracellular polymeric substances (EPS), as microbial high-molecular organic matter widely distributed in the environment, play an important role in environmental ecological processes. However, the study of EPS faces many challenges due to the lack of markers and the difficulty of purification.

[0003] Existing research methods mainly focus on using 13 C. 18 O and 15 Isotopes such as N indirectly trace the synthesis rate and carbon utilization efficiency of environmental microbial cells themselves. During the experimental analysis, researchers added isotope-labeled substrates, such as 13 C and 15 N-labeled organic matter, and 18 The target sample is cultured with HO labeled with HO. After the culture is complete, the biomass of the newly synthesized cells is inferred by precisely measuring changes in DNA content and isotope abundance in the sample. While this method can provide a certain degree of insight into microbial cell characteristics, it is limited in its ability to study the synthesis and metabolism of EPS itself, preventing direct and accurate analysis of EPS synthesis and nutrient utilization.

[0004] Existing methods for studying EPS have obvious shortcomings. First, existing methods cannot directly study EPS and can only indirectly infer it through isotope tracing of microbial cells, making it difficult to accurately focus on the anabolic pathway of EPS itself; second, existing methods use changes in DNA content and isotope abundance to measure the biomass of newly synthesized cells, and lack targeted analysis of EPS-specific components. EPS's polysaccharides, proteins and other components are its core components. Existing methods ignore these key parts, resulting in insufficient accuracy in EPS research; third, existing methods do not involve the separation and purification of EPS, and cannot remove impurities that may interfere with research results. When faced with EPS with complex structures and lack of markers, it is difficult to obtain accurate synthesis rate and carbon utilization efficiency data. Summary of the Invention

[0005] The present invention uses the same isotope treatment method as that used for microbial cell analysis, takes purified polysaccharides and proteins as markers of the total amount of extracellular polymers, and utilizes the previously constructed environmental purification method for polysaccharides and proteins to separate and purify the extracellular polymers after culture. The carbon synthesis rate of the extracellular polymers is quantified by analyzing the content and isotope abundance of the purified polysaccharides and proteins.

[0006] To solve the above problems, the present invention provides a method for analyzing the carbon synthesis rate and carbon utilization efficiency of extracellular polymers, comprising the following steps: S1: Pre-cultivation of sampled soil; S2: Add isotope with a purity of 98.0 at% 18 O-H2O, making the soil 18 The O-H2O content was 20 at% as the labeling group; at the same time, no isotope was added. 18 O-H2O control group; the labeled group and control group were cultured for 24 hours; S3: Using a cation exchange resin method to extract extracellular polymers from the soil of the labeled group and the control group in step S2, and obtaining purified polysaccharides and proteins after separation and purification; S4: Collect the gas produced during the incubation in step S2 and measure the CO2 concentration by gas chromatograph; extract soil DNA, measure the DNA concentration, and measure the DNA content in the DNA sample by isotope mass spectrometry. 18 O abundance and total oxygen content; S5: Determination of the content of purified polysaccharides and proteins by isotope mass spectrometry 18 The abundance and total oxygen content of O were measured to obtain the atomic percentage excess of purified polysaccharides and proteins, respectively; S6: Obtaining the yield of the purified polysaccharide and protein based on the average oxygen content percentage of the purified polysaccharide and protein, the total oxygen content of the purified polysaccharide and protein calculated in step S5, and the ratio of the total amount of the newly produced purified polysaccharide and protein to the newly produced portion of the purified polysaccharide and protein using the oxygen element derived from HO; S7: Obtain the synthesis rate and carbon utilization efficiency of extracellular polymers based on the purified polysaccharide, purified protein, carbon conversion coefficient, and the yields of polysaccharide and protein obtained in step S6.

[0007] Optionally, step S1 specifically includes: mixing the collected soil, removing the root litter part, and storing it in an environment of 4°C after sieving; then, pre-culturing the soil at 15°C and WHC 50% for 7 days.

[0008] Optionally, step S3 specifically includes: weighing 3 g of dry weight of soil, adding 30 mL of 0.01 mol / L, 4°C, pH 7 calcium chloride solution, shaking at 4°C and 120 r / min for 1 hour, centrifuging at 3200 × g for 30 minutes, discarding the supernatant, and then adding 30 mL of 4°C phosphate buffer and cation exchange resin; shaking at 4°C and 180 r / min for 2 hours, centrifuging at 4000 × g for 30 min, and filtering with a 0.45 μm filter membrane; the supernatant is dialyzed and freeze-dried to obtain purified polysaccharide, and the precipitate is washed with acetone, hydrolyzed with urea, and desalted to obtain purified protein.

[0009] Optionally, step S4 further includes: obtaining the soil microbial intracellular carbon synthesis rate and the soil microbial respiration rate.

[0010] Optionally, in step S4: The calculation formula for the soil microbial intracellular carbon synthesis rate is:

[0011] Among them, f DNA It is the ratio of soil microbial carbon content to soil DNA content. produced is the yield of double-stranded DNA during the incubation process. DW is the dry weight of the soil used in step S4, and t is the incubation time in step S2.

[0012] The calculation formula of the soil microbial respiration rate is:

[0013] in, (ppm) is the amount of CO2 generated during the incubation period, M is the molecular mass of C, V is the volume of the headspace bottle, DW is the dry weight of the soil used in step S2, t is the incubation time in step S2; 22.4 is the volume per mole of gas at standard atmospheric pressure.

[0014] Optionally, in step S5, the isotope mass spectrometer is used to determine the 18 The abundance and total oxygen content of O were determined by adding purified water to the purified polysaccharide and protein samples, drying them at 50°C for 6 hours, and measuring the oxygen abundance and total oxygen content using an elemental analyzer and an isotope ratio mass spectrometer. 18 O abundance and total oxygen content.

[0015] Optionally, in step S5, the atomic percentage of the purified polysaccharide and protein exceeds The calculation process is:

[0016] in: It is an atomic percent super, is the abundance of polysaccharide or protein in the labeled sample, is the sample abundance of polysaccharide or protein in the control group.

[0017] Optionally, in step S6, the average oxygen content percentages of the purified polysaccharide and protein are selected to be 29.6% and 38.0%, respectively.

[0018] Optionally, in step S6, the ratio of the total amount of newly produced purified polysaccharides and proteins to the newly produced portion of oxygen element using H2O as a source is obtained by: The soil microbial flora contains 18 After culturing the soil extract of O-H2O in LB medium for different time periods, the extracellular polymers were extracted using the same cation exchange resin method as in step S3; the changes in the content of purified polysaccharides and proteins in the extracellular polymers before and after culturing were analyzed by chemical colorimetry, which was the total amount of newly produced purified polysaccharides and proteins; the purified polysaccharides and proteins were separated and purified in the same manner as in step S3, and the total oxygen content of the purified polysaccharides and proteins was measured by isotope mass spectrometry, and the newly produced purified polysaccharides and proteins using the oxygen element derived from H2O were calculated according to the following formula:

[0019]

[0020] in: and They represent the newly produced polysaccharides and proteins using oxygen from H2O; and are the total oxygen contents of the purified polysaccharide and protein in step S6, respectively; is the final soil solution of the sample 18 O abundance, which is the abundance of O in the soil in step S2 18 O-H2O content 20 at%.

[0021] By fitting the curve between the total amount of the newly produced purified polysaccharides and proteins and the newly produced purified polysaccharides and proteins using the oxygen element from HO, the ratio of the total amount of the newly produced purified polysaccharides and proteins to the newly produced purified polysaccharides and proteins using the oxygen element from HO is determined as a. Poly and a Prot .

[0022] Optionally, in step S7, the calculation formula for the extracellular polymer carbon synthesis rate is as follows:

[0023] Wherein: 0.40, 0.53 and 0.74 are the purified polysaccharide, purified protein and carbon conversion coefficients, respectively; DW is the dry weight of the soil weighed in step S3, and t is the incubation time in step S2; The calculation formula for the carbon utilization efficiency of the extracellular polymeric substances is:

[0024] Among them: CUE EPS represents the carbon utilization efficiency of extracellular polymers, C respiration represents the soil microbial respiration rate, C growth represents the carbon synthesis rate of soil microorganisms; C EPS represents the rate of soil microbial carbon synthesis.

[0025] The present invention's analytical method for extracellular polymer carbon synthesis rate and carbon utilization efficiency has the following beneficial effects: through isotope labeling and control experimental design, it accurately separates the influence of environmental variables, ensuring the reliability of the results; and combines efficient extraction and purification techniques with isotope mass spectrometry to ensure analytical accuracy throughout the entire process, from sample processing to data acquisition. Furthermore, the present invention innovatively constructs a multi-parameter calculation system that not only quantifies the extracellular polymer carbon synthesis rate but also deeply analyzes carbon utilization efficiency and systematically reveals microbial metabolic characteristics, providing a scientific, comprehensive, precise, and efficient research method for soil microbial ecology research, ecosystem function assessment, and agricultural environmental applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The present invention is a flow chart of a method for analyzing the carbon synthesis rate and carbon utilization efficiency of extracellular polymers.

[0027] Figure 2 Schematic diagram showing the relationship between the total amount of newly produced purified polysaccharides of the present invention and the newly produced portion of oxygen element using H2O as a source.

[0028] Figure 3 Schematic diagram showing the relationship between the total amount of newly produced purified protein of the present invention and the ratio of the newly produced portion of oxygen element using H2O as a source. DETAILED DESCRIPTION

[0029] To make the above-mentioned objects, features, and advantages of the present invention more clearly understood, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. Although certain embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be construed as being limited to the embodiments described herein. Instead, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the drawings and embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0030] As used herein, the term "including" and its variations are open-ended, i.e., "including but not limited to"; the term "based on" means "based at least in part on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments"; and the term "optionally" means "optional embodiments." Definitions of other terms are provided in the following description.

[0031] like Figure 1 As shown, an embodiment of the present invention provides a method for analyzing the carbon synthesis rate and carbon utilization efficiency of extracellular polymers, comprising the following steps: S1: Pre-cultivation of sampled soil; S2: Add isotope with a purity of 98.0 at% 18 O-H2O, making the soil 18 The O-H2O content was 20 at% as the labeling group; at the same time, no isotope was added. 18 O-H2O control group; the labeled group and control group were cultured for 24 h; S3: Using a cation exchange resin method to extract extracellular polymers from the soil of the labeled group and the control group in step S2, and obtaining purified polysaccharides and proteins after separation and purification; S4: The gas produced during the incubation in step S2 was collected and the CO2 concentration was measured by gas chromatography. 0.25 g of soil was extracted using the Qiagen DNeasy PowerSoil Kit and the DNA concentration was measured using the Quant-iT PicoGreendsDNA Assay Kit (Invitrogen). The DNA content in the DNA sample was determined by isotope mass spectrometry. 18 O abundance and total oxygen content.

[0032] S5: Determination of the content of purified polysaccharides and proteins by isotope mass spectrometry 18 The abundance and total oxygen content of O were measured to obtain the atomic percentage excess of purified polysaccharides and proteins, respectively; S6: Obtaining the yield of the purified polysaccharide and protein based on the average oxygen content percentage of the purified polysaccharide and protein, the total oxygen content of the purified polysaccharide and protein calculated in step S5, and the ratio of the total amount of the newly produced purified polysaccharide and protein to the newly produced portion of the purified polysaccharide and protein using the oxygen element derived from HO; S7: Obtain the carbon synthesis rate and carbon utilization efficiency of extracellular polymers based on the purified polysaccharide, purified protein, carbon conversion coefficient, and the yields of polysaccharide and protein obtained in step S6.

[0033] In this example, the metabolic state of soil microorganisms was stabilized by 7 days of pre-cultivation, which reduced the data fluctuation caused by environmental mutations and ensured the authenticity and reliability of the experimental data. The setting of the labeled group and the control group can effectively eliminate the interference of non-isotopic factors such as temperature and humidity, and accurately analyze the 18 The influence of O−H2O on synthesis; cation exchange resin extraction combined with separation and purification to avoid interference of impurities on determination and improve data accuracy; comprehensive determination 18 The system builds a complete calculation system based on multiple parameters such as O abundance and total oxygen content, breaking through the limitations of traditional single indicators. It quantifies the synthesis of extracellular polymers from multiple dimensions and deeply reveals the relationship between its synthesis and nutrient utilization, providing rich and accurate data support for studying the metabolic mechanism of soil microorganisms and their impact on the ecological environment.

[0034] Specifically, step S1 includes: collecting and mixing the collected soil, removing root litter, sieving it (2 mm), and storing it at 4°C. The soil is then pre-incubated at 15°C and a WHC of 50% for 7 days. In this invention, the soil sample collected is Heilongjiang forest soil (black soil).

[0035] In this example, soil samples were pre-cultured for 7 days to allow the soil microorganisms to fully adapt to the experimental environment and stabilize their community activity and metabolic state. In a stable state, microorganisms have more regular metabolic activities, avoiding metabolic disturbances caused by environmental changes, thereby reducing data fluctuations. In subsequent steps, analysis is performed based on the extracellular polymers synthesized by microorganisms in this stable state, making the measured carbon synthesis rate and carbon utilization efficiency data more realistically reflect the natural state, significantly improving reliability and accuracy.

[0036] Specifically, step S3 specifically includes: weighing about 3 g of soil (dry weight) in a 50 mL centrifuge tube, adding 30 mL of 0.01 mol / L, 4°C calcium chloride solution (pH 7), shaking on a shaker at 4°C and 120 r / min for 1 hour, centrifuging at 3200 × g for 30 minutes, discarding the supernatant, and then adding 30 mL of 4°C phosphate buffer and cation exchange (CER) resin (the content of CER added to the soil is 178 mg CER mg -1 The mixture was then shaken at 4°C and 180 rpm for 2 hours, centrifuged at 4000 × g for 30 minutes, and filtered through a 0.45 μm filter membrane. The supernatant was dialyzed and freeze-dried to obtain purified polysaccharides, and the precipitate was washed with acetone, hydrolyzed with urea, and desalted to obtain purified proteins.

[0037] The phosphate buffer solution consisted of 2 mM trisodium phosphate dodecahydrate, 4 mM sodium dihydrogen phosphate dihydrate, 9 mM sodium chloride, and 1 mM potassium chloride, and was adjusted to pH 7.0 with 1 M hydrochloric acid and cooled to 4 °C.

[0038] In this example, extracellular polymeric substances (ECPs) were extracted from soil samples from the labeled and control groups in step S2 using a cation exchange resin method. This method effectively separates ECPs from soil, avoiding interference from other soil components. The extracted ECPs then undergo a series of separation and purification steps to remove impurities, ultimately yielding purified polysaccharides and proteins, providing a pure sample for subsequent accurate determination of their isotopic abundance and content.

[0039] Preferably, step S4 further includes: obtaining the soil microbial intracellular carbon synthesis rate and the soil microbial respiration rate.

[0040] Specifically, the calculation formula for the intracellular carbon synthesis rate of soil microorganisms is:

[0041] Among them, f DNA It is the ratio of soil microbial carbon content to soil DNA content. produced is the yield of double-stranded DNA during the incubation process. DW (g) is the dry weight of the soil used in step S4, and t (h) is the incubation time in step S2.

[0042] The calculation formula for soil microbial respiration rate is:

[0043] in, (ppm) is the amount of CO2 produced during the culture period, M (12.01 g mol - 1 ) is the molecular weight of C, V (L) is the volume of the headspace bottle, DW (g) is the dry weight of the soil used in S2, and t (h) is the incubation time in step S2; 22.4 ( L mol -1 ) is the volume per mole of gas at standard atmospheric pressure.

[0044] Among them, the soil DNA content was determined by the following method: soil DNA was extracted using a kit (Qiagen DNeasyPowerSoil Kit), and the soil DNA content was determined by Pico-green staining after DNA extraction.

[0045] Soil microbial biomass carbon (MBC) content was determined as follows: 6 g dry weight equivalent soil was weighed, 3 g of which was placed in a 10 mL beaker and placed in a vacuum desiccator. The desiccator was then fumigated for 24 hours in the dark at 25°C with 50 mL of ethanol-free chloroform and 50 mL of 1 mol / L sodium hydroxide. This served as the fumigation treatment. The remaining 3 g dry weight equivalent soil was placed in a centrifuge tube and kept in the dark at 25°C for 24 hours, serving as the unfumigated control. Subsequently, 12 mL of 0.5 mol / L K₂SO₄ solution was added to all treatments, shaken at 180 rpm for 1 hour, centrifuged at 5000 × g for 5 minutes, and filtered through a 0.45 μm filter. The supernatant was diluted 10-fold and carbon content was determined using a total organic carbon analyzer. The difference in soluble organic carbon content between the extracts of the fumigated sample and the corresponding unfumigated sample was divided by the conversion coefficient of 0.45 to obtain the soil microbial biomass carbon content.

[0046] The yield of double-stranded DNA during the incubation period was calculated according to the following formula:

[0047] in, is the total oxygen content of the extracted DNA (μg), and the oxygen mass of DNA accounts for 31.21%. is the atomic percentage excess, which is the amount of DNA sample labeled in step S4. 18 O abundance relative to natural abundance samples 18 The difference in the average abundance of O. is the final soil solution of the sample 18 O abundance, i.e., the abundance of O in the soil in step S2 18 O-H2O content 20 at%.

[0048] In this example, the intracellular carbon synthesis rate of soil microorganisms and the soil microbial respiration rate are closely related to carbon utilization efficiency. By analyzing them, we can deeply explore how microorganisms coordinate physiological processes such as growth, metabolism and extracellular polymer synthesis, and provide a theoretical basis for understanding the ecological functions of microorganisms.

[0049] Specifically, in step S5, the isotope mass spectrometer is used to determine the concentration of purified polysaccharides and protein samples. 18 The abundance and total oxygen content of O were determined by adding purified water to the purified polysaccharide and protein samples, drying them at 50°C for 6 h, and measuring them using an elemental analyzer and an isotope ratio mass spectrometer. 18 O abundance and total oxygen content.

[0050] Specifically, the atomic percent of purified polysaccharides and proteins exceeds The calculation process is: ; in: It is an atomic percent super, is the abundance of polysaccharide or protein in the labeled group sample, i.e., the abundance of polysaccharide or protein measured on the machine in step S5. 18 O abundance, is the sample abundance of polysaccharide or protein in the control group.

[0051] In this embodiment, the purified polysaccharide and protein samples were detected by isotope mass spectrometry to determine the 18 The abundance of O−H2O and the total oxygen content. Based on the measured data, the atomic percentage excess of the purified polysaccharide and protein was calculated. The calculation of atomic percentage excess can reflect the enrichment of isotope labels in polysaccharides and proteins, and thus understand the utilization of microorganisms. 18 O−H2O synthesis of extracellular polymers.

[0052] Specifically, the ratio of the total amount of newly produced purified polysaccharides and proteins to the newly produced portion of oxygen element using H2O is as follows: The soil microbial flora contains 18 After incubation in LB medium with the soil extract of O-H2O for different time periods, the extracellular polymers were extracted using the same cation exchange resin method as in step S3 above. The changes in the content of purified polysaccharides and proteins in the extracellular polymers before and after incubation were analyzed by chemical colorimetry, which was the total amount of newly produced purified polysaccharides and proteins. The purified polysaccharides and proteins were separated and purified in the same manner as in step S3 above. The total oxygen content of the purified polysaccharides and proteins was measured by isotope mass spectrometry, and the newly produced purified polysaccharides and proteins using the oxygen element derived from H2O were calculated according to the following formula:

[0053]

[0054] in: and They represent the newly produced polysaccharides and proteins using oxygen from H2O; and are the total oxygen contents of the purified polysaccharide and protein in step S5, respectively. is the final soil solution of the sample 18 O abundance, i.e., the abundance of O in the soil in step S2 18 O-H2O content 20 at%.

[0055] Soil microbial flora is obtained by mixing soil with sterile buffer and shaking to form a suspension, and then separating microbial cells and soil particles by density gradient centrifugation.

[0056] Specifically, the average oxygen content percentages of the purified polysaccharide and protein were selected as 29.6% and 38.0%, respectively, which were specifically calculated based on all samples.

[0057] By fitting the curve between the total amount of the newly produced purified polysaccharides and proteins and the newly produced purified polysaccharides and proteins using the oxygen element from HO, the ratio of the total amount of the newly produced purified polysaccharides and proteins to the newly produced purified polysaccharides and proteins using the oxygen element from HO is determined as a. Poly and a Prot .

[0058] like Figure 2 and 3 As shown, the ratio of the total amount of newly produced purified polysaccharides and proteins to the newly produced purified polysaccharides and proteins using the oxygen element of H2O is shown, that is, a Poly and a Prot They are 6.05 and 6.26 respectively. In the figure, the X-axis is the newly produced purified polysaccharide or protein using the oxygen element from H2O, and the Y-axis is the total amount of the newly produced purified polysaccharide or protein.

[0059] In this example, the yield of purified polysaccharides and proteins was accurately calculated using a specific formula based on the average oxygen content percentage of the purified polysaccharides and proteins, combined with the total oxygen content of the purified polysaccharides and proteins calculated in step S5, and the ratio between the total amount of newly produced purified polysaccharides and proteins and the newly produced oxygen from HO. These parameters are interrelated and reflect the oxygen utilization during extracellular polymer synthesis from different perspectives, thereby inferring the actual production of polysaccharides and proteins.

[0060] Specifically, in step S7, the calculation formula for the carbon synthesis rate of extracellular polymers is as follows:

[0061] Wherein: 0.40, 0.53 and 0.74 are the carbon conversion coefficients of purified polysaccharide, purified protein and purified carbon, respectively; DW is the dry weight of the soil weighed in step S3; and t is the incubation time in step S2.

[0062] The carbon utilization efficiency of extracellular polymers is calculated as follows:

[0063] Among them: CUE EPS represents the carbon utilization efficiency of extracellular polymers, Crespiration represents the carbon flux allocated to respiration (ng C g −1 h −1 ), C growth represents the carbon synthesis rate of soil microorganisms (ng C g −1 h −1 );C EPS represents the extracellular carbon synthesis rate of soil microorganisms (ng C g −1 h −1 ).

[0064] In this example, carbon utilization efficiency is a key indicator for analyzing microbial metabolic strategies. Based on the carbon conversion coefficients between purified polysaccharides, purified proteins, and purified carbon, combined with the polysaccharide and protein yields calculated in step S6, the carbon synthesis rate of extracellular polymeric substances (ECPs) is ultimately calculated using appropriate mathematical models and calculation methods. The carbon conversion coefficient reflects the conversion relationship between polysaccharides, proteins, and carbon. This coefficient can be used to convert polysaccharide and protein yields into the overall carbon synthesis rate of ECPs, providing a direct reflection of the efficiency of ECP synthesis by the microorganism.

[0065] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for analyzing the carbon synthesis rate and carbon utilization efficiency of extracellular polymers, characterized in that: The following steps are involved: S1: Pre-cultivation of sampled soil; S2: Add isotope with a purity of 98.0 at% 18 O-H2O, making the soil 18 The O-H2O content was 20 at% as the labeling group; at the same time, no isotope was added. 18 O-H2O control group; the labeled group and control group were cultured for 24 hours; S3: Using a cation exchange resin method to extract extracellular polymers from the soil of the labeled group and the control group in step S2, and obtaining purified polysaccharides and proteins after separation and purification; S4: Collect the gas generated during the incubation in step S2 and measure the CO2 concentration by gas chromatograph; extract soil DNA, measure the DNA concentration, and measure the DNA content in the DNA sample by isotope mass spectrometry. 18 O abundance and total oxygen content; S5: Determination of the content of purified polysaccharides and proteins by isotope mass spectrometry 18 The abundance and total oxygen content of O were measured to obtain the atomic percentage excess of purified polysaccharides and proteins, respectively; S6: Obtaining the yield of the purified polysaccharide and protein based on the average oxygen content percentage of the purified polysaccharide and protein, the total oxygen content of the purified polysaccharide and protein calculated in step S5, and the ratio of the total amount of the newly produced purified polysaccharide and protein to the newly produced portion of the purified polysaccharide and protein using the oxygen element derived from HO; S7: Obtain the carbon synthesis rate and carbon utilization efficiency of extracellular polymers based on the purified polysaccharide, purified protein, carbon conversion coefficient, and the yields of polysaccharide and protein obtained in step S6.

2. The method for analyzing the carbon synthesis rate and carbon utilization efficiency of extracellular polymers according to claim 1, characterized in that: Step S1 includes: The collected soil was mixed, root litter was removed, and it was sieved and stored at 4°C. After that, the soil was pre-incubated at 15°C and WHC 50% for 7 days.

3. The method for analyzing the carbon synthesis rate and carbon utilization efficiency of extracellular polymers according to claim 1, characterized in that: Step S3 includes: Weigh 3 g of dry soil, add 30 mL of 0.01 mol / L, 4°C, pH 7 calcium chloride solution, shake at 4°C, 120 r / min for 1 hour, centrifuge at 3200 × g for 30 minutes, discard the supernatant, and then add 30 mL of 4°C phosphate buffer and cation exchange resin; shake at 4°C, 180 r / min for 2 hours, centrifuge at 4000 × g for 30 minutes, and filter with a 0.45 μm filter membrane; the supernatant is dialyzed and freeze-dried to obtain purified polysaccharides, and the precipitate is washed with acetone, hydrolyzed with urea, and desalted to obtain purified proteins.

4. The method for analyzing the carbon synthesis rate and carbon utilization efficiency of extracellular polymers according to claim 1, characterized in that: Step S4 also includes: obtaining the soil microbial intracellular carbon synthesis rate and the soil microbial respiration rate.

5. The method for analyzing the carbon synthesis rate and carbon utilization efficiency of extracellular polymers according to claim 4, characterized in that: In step S4: The calculation formula for the intracellular carbon synthesis rate of soil microorganisms is: Among them, f DNA It is the ratio of soil microbial carbon content to soil DNA content. produced is the yield of double-stranded DNA during the incubation process, DW is the dry weight of the soil used in step S4, and t is the incubation time in step S2; The calculation formula for soil microbial respiration rate is: in, is the amount of CO2 generated during the incubation period, M is the molecular mass of C, V is the volume of the headspace bottle, DW is the dry weight of the soil used in step S2, t is the incubation time in step S2; 22.4 is the volume per mole of gas at standard atmospheric pressure.

6. The method for analyzing the carbon synthesis rate and carbon utilization efficiency of extracellular polymers according to claim 1, characterized in that: In step S5, the isotope mass spectrometer is used to determine the content of purified polysaccharide and protein samples. 18 O abundance and total oxygen content, including: The purified polysaccharide and protein samples were dissolved in pure water, dried at 50°C for 6 hours, and analyzed using an elemental analyzer and an isotope ratio mass spectrometer. 18 O abundance and total oxygen content.

7. The method for analyzing the carbon synthesis rate and carbon utilization efficiency of extracellular polymers according to claim 1, characterized in that: In step S5, the atomic percentage excess of the purified polysaccharide and protein is calculated as follows: ； in: It is an atomic percent super, is the abundance of polysaccharide or protein in the labeled sample group, is the polysaccharide or protein abundance of the control sample.

8. The method for analyzing extracellular polymer carbon synthesis rate and carbon utilization efficiency according to claim 1, characterized in that: In step S6, the average oxygen content percentages of the purified polysaccharide and protein are selected as 29.6% and 38.0%, respectively.

9. The method for analyzing the carbon synthesis rate and carbon utilization efficiency of extracellular polymers according to claim 8, characterized in that: In step S6, the ratio of the total amount of newly produced purified polysaccharides and proteins to the newly produced portion of oxygen element using H2O is obtained as follows: The soil microbial flora contains 18 After incubation in soil extracts of O-H2O and LB medium for different periods of time, extracellular polymers were extracted using a cation exchange resin method. The changes in the content of purified polysaccharides and proteins in the extracellular polymers before and after incubation were analyzed by chemical colorimetry, which was the total amount of newly produced purified polysaccharides and proteins. The purified polysaccharides and proteins were separated and purified, and the purified polysaccharides and proteins were determined by isotope mass spectrometry. 18 O abundance and total oxygen content were calculated, and the newly produced purified polysaccharides and proteins using HO-derived oxygen were calculated according to the following formula: in: and They represent the newly produced polysaccharides and proteins using oxygen from H2O; and are the total oxygen contents of the purified polysaccharide and protein in step S6, respectively; is the final soil solution of the sample 18 O abundance, which is the abundance of O in the soil in step S2 18 O-H2O content 20 at%. By fitting the curve between the total amount of the newly produced purified polysaccharides and proteins and the newly produced purified polysaccharides and proteins using the oxygen element from HO, the ratio of the total amount of the newly produced purified polysaccharides and proteins to the newly produced purified polysaccharides and proteins using the oxygen element from HO is determined as a. Poly and a Prot .

10. The method for analyzing extracellular polymer carbon synthesis rate and carbon utilization efficiency according to claim 9, characterized in that: In step S7, the calculation formula for the carbon synthesis rate of extracellular polymers is as follows: Wherein: 0.40, 0.53 and 0.74 are the purified polysaccharide, purified protein and carbon conversion coefficients, respectively; DW is the dry weight of the soil weighed in step S3, and t is the incubation time in step S2; The calculation formula for the carbon utilization efficiency of the extracellular polymeric substances is: Among them: CUE EPS represents the carbon utilization efficiency of extracellular polymers, C respiration represents the soil microbial respiration rate, C growth represents the carbon synthesis rate of soil microorganisms; C EPS Represents the rate of extracellular carbon synthesis by soil microorganisms.

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