An analysis method for extracellular polymeric substance carbon synthesis rate and carbon utilization efficiency

Abstract

This invention provides an analytical method for the extracellular polymeric carbon synthesis rate and carbon use efficiency, relating to the technical field of analytical testing. The method includes: pre-culturing sampled soil; adding an isotope with a purity of 98.0 at% 18 O-H2O, making the soil 18 A marker group was defined as having an O-H2O content of 20 at%; a group without added isotopes was also included. 18 The control group was treated with O-H2O. Extracellular polymers were extracted from the soil of both the labeled and control groups using a cation exchange resin method. After separation and purification, purified polysaccharides and proteins were obtained. Isotope mass spectrometry was used to determine the concentrations of O-H2O in the purified polysaccharide and protein samples. 18 The abundance of O and total oxygen content were used to calculate the atomic percentage of purified polysaccharides and proteins, respectively; the yield of purified polysaccharides and proteins was calculated; and the carbon synthesis rate and carbon utilization efficiency of extracellular polymers were calculated.

Description

Technical Field

[0001] This invention relates to the technical field of analytical testing, and more specifically, to an analytical method for the extracellular polymeric carbon synthesis rate and carbon utilization efficiency. Background Technology

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

[0003] Existing research methods mainly focus on utilizing 13 C 18 O and 15 Nitrogen isotopes were used to indirectly trace the synthesis rate and carbon utilization efficiency of environmental microbial cells. During the experimental analysis, researchers added isotope-labeled substrates, such as... 13 C and 15 N-labeled organic matter, and 18 O-labeled H2O was used to culture the target sample. After culturing, the biomass of newly synthesized cells was inferred by precisely detecting changes in DNA content and isotope abundance in the sample. While this method can explore the characteristics of microbial cells to some extent, it still has limitations in studying the synthesis and metabolism of EPS itself, and cannot directly and accurately analyze the synthesis and nutrient utilization of EPS.

[0004] Current methods for studying EPS have significant shortcomings. First, they cannot directly study EPS, but can only indirectly infer its synthesis through isotope tracing of microbial cells, making it difficult to accurately focus on the synthetic metabolic pathways of EPS itself. Second, current methods measure the biomass of newly synthesized cells by changes in DNA content and isotope abundance, lacking targeted analysis of EPS-specific components. However, the polysaccharides and proteins of EPS are its core components, and current methods ignore these key parts, resulting in insufficient precision in EPS research. Third, current methods do not involve the isolation and purification of EPS, failing to remove impurities that may interfere with the research results. When dealing with structurally complex EPS that lacks markers, it is difficult to obtain accurate data on synthesis rate and carbon utilization efficiency. Summary of the Invention

[0005] This invention employs the same isotope treatment method as microbial cell analysis, using purified polysaccharides and proteins as markers of total extracellular polymeric substances (APIs). The cultured APIs are separated and purified using a previously constructed environmental purification method for polysaccharides and proteins. The carbon synthesis rate of APIs is quantified by analyzing the content of purified polysaccharides and proteins and their isotope abundance.

[0006] To address the above problems, this invention provides a method for analyzing the extracellular polymeric carbon synthesis rate and carbon utilization efficiency, comprising the following steps:

[0007] S1: Pre-culture the sampled soil;

[0008] S2: Add an isotope with a purity of 98.0 at%. 18 O-H2O, making the soil 18 A marker group was defined as having an O-H2O content of 20 at%; a group without added isotopes was also included. 18 O-H2O control group; the labeled group and the control group were cultured for 24 hours;

[0009] S3: The extracellular polymers in the soil of the labeled group and the control group in step S2 were extracted by cation exchange resin method, and purified polysaccharides and proteins were obtained after separation and purification.

[0010] S4: Collect the gases generated during the cultivation process in step S2 and determine the CO2 concentration using a gas chromatograph; extract soil DNA, determine the DNA concentration, and analyze the DNA sample using an isotope mass spectrometer. 18 O abundance and total oxygen content;

[0011] S5: Determination of purified polysaccharide and protein samples using isotope mass spectrometry. 18 The abundance of O and total oxygen content were used to obtain the atomic percentage of purified polysaccharides and proteins, respectively.

[0012] S6: Based on the average oxygen content percentage of the purified polysaccharides and proteins, the total oxygen content of the purified polysaccharides and proteins calculated in step S5, and the ratio of the total amount of newly generated purified polysaccharides and proteins to the newly generated portion of purified polysaccharides and proteins using oxygen elements from H2O, obtain the yield of purified polysaccharides and proteins.

[0013] S7: Based on the purified polysaccharide, purified protein, carbon conversion coefficient, and the yield of polysaccharide and protein obtained in step S6, obtain the extracellular polymer synthesis rate and carbon utilization efficiency.

[0014] Optionally, step S1 specifically includes: mixing the collected soil, removing root litter, sieving and storing it in an environment of 4°C; then, pre-culturing the soil at 15°C and 50% WHC for 7 days.

[0015] Optionally, step S3 specifically includes: weighing 3g of dry 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, 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 minutes, and filtering with a 0.45 μm filter membrane; the supernatant is dialyzed and lyophilized to obtain purified polysaccharide, and the precipitate is washed with acetone, hydrolyzed with urea, and desalted with peptides to obtain purified protein.

[0016] Optionally, step S4 may further include: obtaining the intracellular carbon synthesis rate and the respiration rate of soil microorganisms.

[0017] Optionally, in step S4:

[0018] The formula for calculating the intracellular carbon synthesis rate of soil microorganisms is as follows:

[0019]

[0020] Among them, f DNA It is the ratio of soil microbial biomass carbon content to soil DNA content. produced This represents the yield of double-stranded DNA during incubation. DW is the dry weight of the soil used in step S4, and t is the incubation time in step S2.

[0021] The formula for calculating the soil microbial respiration rate is as follows:

[0022]

[0023] in, (ppm) represents the amount of CO2 generated during the incubation period, M is the molecular weight of C, V is the volume of the headspace vial, DW is the dry weight of the soil used in step S2, and t is the incubation time in step S2; 22.4 is the volume of one mole of gas under standard atmospheric pressure.

[0024] Optionally, in step S5, the determination of the content of purified polysaccharides and proteins in the sample using isotope mass spectrometry... 18 The abundance of O and total oxygen content were determined by dissolving the purified polysaccharide and protein samples in pure water, drying them at 50°C for 6 hours, and measuring them using an elemental analyzer and isotope ratio mass spectrometer. 18 O abundance and total oxygen content.

[0025] Optionally, in step S5, the atomic percentage of the purified polysaccharides and proteins is ultrapure. The calculation process is as follows:

[0026]

[0027] in: It is atomic percentage over, It refers to the abundance of polysaccharides or proteins in the labeled sample. This represents the abundance of polysaccharides or proteins in the control group samples.

[0028] Optionally, in step S6, the average oxygen content percentages of the purified polysaccharides and proteins are selected as 29.6% and 38.0%, respectively.

[0029] Optionally, in step S6, the process for obtaining the ratio of the total amount of newly generated purified polysaccharides and proteins to the newly generated portion using oxygen from H2O is as follows:

[0030] Soil microbial communities containing 18 After culturing the soil extract containing O-H2O in LB medium for different time periods, 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 culture were analyzed by a chemical colorimetric method to determine the total amount of newly generated purified polysaccharides and proteins. The purified polysaccharides and proteins were then separated and purified using the same method as in step S3. The total oxygen content of the purified polysaccharides and proteins was determined by isotope mass spectrometry, and the amount of newly generated purified polysaccharides and proteins using oxygen from H2O was calculated according to the following formula:

[0031]

[0032]

[0033] in: and These represent the polysaccharides and proteins newly generated using oxygen from H2O, respectively. and These are the total oxygen content of the purified polysaccharides and proteins in step S6; It is in the final soil solution of the sample 18 O abundance, which is the abundance of soil in step S2. 18 O-H2O content 20 at%.

[0034] By fitting a curve between the total amount of newly generated purified polysaccharides and proteins and the newly generated purified polysaccharides and proteins using oxygen from H2O, the ratio of the total amount of newly generated purified polysaccharides and proteins to the newly generated purified polysaccharides and proteins using oxygen from H2O is determined to be a. Poly and a Prot .

[0035] Optionally, in step S7, the formula for calculating the extracellular polymeric carbon synthesis rate is as follows:

[0036]

[0037] Where: 0.40, 0.53 and 0.74 are the carbon conversion coefficients of purified polysaccharide, purified protein and carbon, respectively; DW is the dry weight of soil weighed in step S3 and t is the culture time in step S2;

[0038] The formula for calculating the carbon utilization efficiency of the extracellular polymeric material is as follows:

[0039]

[0040] Among them: CUE EPS C represents the carbon utilization efficiency of extracellular polymers. respiration C represents the soil microbial respiration rate. growth Represents the rate of intracellular carbon synthesis in soil microorganisms; C EPS This represents the rate of soil microbial carbon synthesis.

[0041] The beneficial effects of the analytical method for extracellular polymeric carbon synthesis rate and carbon use efficiency proposed in this invention are as follows: Through isotope labeling and control experimental design, the influence of environmental variables is accurately separated, ensuring the reliability of the results; combined with efficient extraction and purification technology and isotope mass spectrometry, the entire process from sample processing to data acquisition guarantees analytical accuracy. Furthermore, this invention innovatively constructs a multi-parameter calculation system, which not only quantifies the extracellular polymeric carbon synthesis rate but also deeply analyzes carbon use efficiency, systematically revealing the characteristics of microbial metabolism. This provides a scientific, comprehensive, accurate, and efficient research method for soil microbial ecology research, ecosystem function assessment, and agricultural environmental applications. Attached Figure Description

[0042] Figure 1 This is a flowchart of a method for analyzing the extracellular polymeric carbon synthesis rate and carbon utilization efficiency according to the present invention.

[0043] Figure 2 This is a schematic diagram showing the ratio between the total amount of newly purified polysaccharides produced in this invention and the newly generated portion using oxygen from H2O.

[0044] Figure 3 This is a schematic diagram showing the ratio of the total amount of newly generated purified protein to the newly generated portion using oxygen from H2O. Detailed Implementation

[0045] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Although some embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the accompanying 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.

[0046] The term "comprising" and its variations as used herein are open-ended inclusion, meaning "including but not limited to"; the term "based on" means "at least partially based 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 will be given in the description below.

[0047] like Figure 1 As shown in the figure, an analytical method for the extracellular polymeric carbon synthesis rate and carbon utilization efficiency provided by an embodiment of the present invention includes the following steps:

[0048] S1: Pre-culture the sampled soil;

[0049] S2: Add an isotope with a purity of 98.0 at%. 18 O-H2O, making the soil 18 A marker group was defined as having an O-H2O content of 20 at%; a group without added isotopes was also included. 18 O-H2O control group; the labeled group and the control group were cultured for 24 h;

[0050] S3: The extracellular polymers in the soil of the labeled group and the control group in step S2 were extracted by cation exchange resin method, and purified polysaccharides and proteins were obtained after separation and purification.

[0051] S4: Collect the gases generated during the cultivation process in step S2, determine the CO2 concentration using gas chromatography, and extract soil DNA from 0.25 g of soil using the Qiagen DNeasy PowerSoil Kit. Determine the DNA concentration using the Quant-iT PicoGreendsDNA Assay Kit (Invitrogen), and analyze the concentration of the DNA sample using isotope mass spectrometry. 18 O abundance and total oxygen content.

[0052] S5: Determination of purified polysaccharide and protein samples using isotope mass spectrometry. 18The abundance of O and total oxygen content were used to obtain the atomic percentage of purified polysaccharides and proteins, respectively.

[0053] S6: Based on the average oxygen content percentage of the purified polysaccharides and proteins, the total oxygen content of the purified polysaccharides and proteins calculated in step S5, and the ratio of the total amount of newly generated purified polysaccharides and proteins to the newly generated portion of purified polysaccharides and proteins using oxygen elements from H2O, obtain the yield of purified polysaccharides and proteins.

[0054] S7: Based on the purified polysaccharide, purified protein, carbon conversion coefficient, and the yield of polysaccharide and protein obtained in step S6, obtain the carbon synthesis rate and carbon utilization efficiency of the extracellular polymeric material.

[0055] In this embodiment, a 7-day pre-culture was used to stabilize the metabolic state of soil microorganisms, reducing data fluctuations caused by sudden environmental changes and ensuring the authenticity and reliability of experimental data. The inclusion of labeled and control groups effectively eliminated interference from non-isotopic factors such as temperature and humidity, enabling precise analysis. 18 The influence of O−H2O on synthesis; extraction and purification using cation exchange resin to avoid interference from impurities and improve data accuracy; comprehensive determination. 18 By constructing a complete calculation system based on multiple parameters such as O abundance and total oxygen content, this approach breaks through the limitations of traditional single indicators, quantifies extracellular polymer synthesis from multiple dimensions, and deeply reveals the relationship between its synthesis and nutrient utilization. This provides rich and accurate data support for studying soil microbial metabolic mechanisms and their impact on the ecological environment.

[0056] Specifically, step S1 includes: mixing the collected soil, removing root litter, sieving (2 mm), and storing it at 4°C; then, pre-culturing the soil at 15°C and 50% WHC for 7 days. In this invention, the collected soil sample is forest soil (black soil) from Heilongjiang Province.

[0057] In this embodiment, soil samples were pre-cultured for 7 days to allow soil microorganisms to fully adapt to the experimental environment and stabilize their community activity and metabolic state. Under stable conditions, microbial metabolic activity is more regular, avoiding metabolic disturbances caused by sudden environmental changes, thus reducing data fluctuations. In subsequent steps, analysis was performed based on extracellular polymers synthesized by microorganisms under stable conditions, making the measured carbon synthesis rate and carbon use efficiency data more accurately reflect the natural state, significantly improving reliability and accuracy.

[0058] Specifically, step S3 includes: weighing approximately 3g of soil (dry weight) into 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 phosphate buffer at 4°C and cation exchange resin (CER) resin (the CER content added to the soil is 178 mg CER mg). -1 SOC). Then, it was shaken at 4 °C and 180 r / min for 2 hours, centrifuged at 4000×g for 30 minutes, and filtered through a 0.45 μm filter membrane; the supernatant was dialyzed and lyophilized to obtain purified polysaccharide, and the precipitate was washed with acetone, hydrolyzed with urea, and desalted by peptide to obtain purified protein.

[0059] The phosphate buffer solution consists of: 2 mM trisodium phosphate dodecahydrate, 4 mM sodium dihydrogen phosphate dihydrate, 9 mM sodium chloride, and 1 mM potassium chloride, adjusted to pH 7.0 with 1 M hydrochloric acid and cooled to 4 °C.

[0060] In this embodiment, the cation exchange resin method was used to extract extracellular polymers from the soil samples of the labeled and control groups in step S2. This method can effectively separate extracellular polymers from the soil and avoid interference from other soil components. The extracted extracellular polymers then undergo a series of separation and purification steps to remove impurities, ultimately obtaining purified polysaccharides and proteins, providing pure samples for subsequent accurate determination of their isotopic abundance and content.

[0061] Preferably, step S4 further includes: obtaining the intracellular carbon synthesis rate and the respiration rate of soil microorganisms.

[0062] Specifically, the formula for calculating the intracellular carbon synthesis rate of soil microorganisms is as follows:

[0063]

[0064] Among them, f DNA It is the ratio of soil microbial biomass carbon content to soil DNA content. produced This represents the yield of double-stranded DNA during incubation. DW (g) is the dry weight of the soil used in step S4, and t (h) is the incubation time in step S2.

[0065] The formula for calculating the soil microbial respiration rate is:

[0066]

[0067] in, (ppm) represents the amount of CO2 generated during the cultivation period, M (12.01 g mol) - 1 ) represents the molecular weight of C, V (L) represents the volume of the headspace vial, DW (g) represents the dry weight of the soil used in S2, and t (h) represents the incubation time in step S2; 22.4 (L mol) -1 () is the volume of one mole of gas under standard atmospheric pressure.

[0068] Soil DNA content was determined as follows: Soil DNA was extracted using a kit (Qiagen DNeasyPowerSoil Kit), and the soil DNA content was determined using the Pico-green staining method after DNA extraction.

[0069] Soil microbial biomass carbon (MBC) content was determined as follows: 6g of dry weight equivalent soil was weighed, and 3g of this dry weight equivalent soil was placed in a 10mL beaker and then placed in a vacuum desiccator. The desiccator was then filled with 50mL of ethanol-free chloroform and 50mL of 1mol / L sodium hydroxide. The mixture was fumigated for 24 hours in the dark at 25°C, serving as the fumigation treatment group. The remaining 3g of dry weight equivalent soil was placed in a centrifuge tube and placed in the dark at 25°C for 24 hours, serving as the non-fumigation control group. Afterwards, 12mL of 0.5mol / L K₂SO₄ solution was added to all treatment groups, and the mixture was shaken at 180 r / min for 1 hour, centrifuged at 5000×g for 5 minutes, filtered through a 0.45 μm filter membrane, and the supernatant was diluted 10-fold before the carbon content was determined using a total organic carbon analyzer. The soil microbial biomass carbon content is obtained by dividing the difference in soluble organic carbon content between the fumigated sample and the corresponding non-fumigated sample extract by a conversion factor of 0.45.

[0070] The yield of double-stranded DNA during incubation is calculated using the following formula:

[0071]

[0072] in, This is the total oxygen content (μg) of the extracted DNA, with oxygen accounting for 31.21% of the DNA mass.

[0073] It refers to atomic percentage, specifically the labeling of DNA samples in step S4. 18 O abundance relative to naturally abundant samples 18 The difference in average abundance of O. It is in the final soil solution of the sample 18 O abundance, i.e., the abundance in the soil during step S2 18 O-H2O content 20 at%.

[0074] In this embodiment, the intracellular carbon synthesis rate and respiration rate of soil microorganisms are closely related to carbon use efficiency. By analyzing them, we can explore in depth 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.

[0075] Specifically, in step S5, isotope mass spectrometry is used to determine the concentration of polysaccharides and proteins in the purified polysaccharide and protein samples. 18 The abundance of O and total oxygen content were determined by dissolving the purified polysaccharide and protein samples in pure water, drying them at 50°C for 6 h, and measuring them using an elemental analyzer and isotope ratio mass spectrometer. 18 O abundance and total oxygen content.

[0076] Specifically, the atomic percentage of purified polysaccharides and proteins exceeded... The calculation process is as follows:

[0077] ;

[0078] in: It is atomic percentage over, It refers to the abundance of polysaccharides or proteins in the labeled sample, i.e., the abundance of polysaccharides or proteins determined by the instrument in step S5. 18 O abundance, This represents the abundance of polysaccharides or proteins in the control group samples.

[0079] In this embodiment, isotope mass spectrometry was used to detect the purified polysaccharide and protein samples and determine their composition. 18 The abundance of O−H2O and total oxygen content were measured. Based on the data, the atomic percentage ultrastructure (AUS) of the purified polysaccharides and proteins was calculated. AUS calculations reflect the enrichment of isotopic labels in polysaccharides and proteins, thus providing insight into microbial utilization. 18 The synthesis of extracellular polymers using O−H2O.

[0080] Specifically, the ratio of the total amount of newly produced purified polysaccharides and proteins to the newly produced portion using oxygen from H2O is as follows:

[0081] Soil microbial communities containing 18After culturing the soil extract containing O-H2O in LB medium for different time periods, 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 culture were analyzed by chemical colorimetric method, which represents the total amount of newly generated purified polysaccharides and proteins. The purified polysaccharides and proteins were then separated and purified in the same way as in step S3 above. The total oxygen content of the purified polysaccharides and proteins was determined by isotope mass spectrometry, and the amount of newly generated purified polysaccharides and proteins using oxygen from H2O was calculated according to the following formula:

[0082]

[0083]

[0084] in: and These represent the polysaccharides and proteins newly generated using oxygen from H2O, respectively. and These refer to the total oxygen content of the purified polysaccharides and proteins in step S5. It is in the final soil solution of the sample 18 O abundance, i.e., the abundance in the soil during step S2 18 O-H2O content 20 at%.

[0085] Soil microbial communities are obtained by mixing soil with sterile buffer solution and shaking to form a suspension, followed by separation of microbial cells and soil particles by density gradient centrifugation.

[0086] Specifically, the average oxygen content percentages of purified polysaccharides and proteins were selected as 29.6% and 38.0%, respectively, which were calculated based on all samples.

[0087] By fitting a curve between the total amount of newly generated purified polysaccharides and proteins and the newly generated purified polysaccharides and proteins using oxygen from H2O, the ratio of the total amount of newly generated purified polysaccharides and proteins to the newly generated purified polysaccharides and proteins using oxygen from H2O is determined to be a. Poly and a Prot .

[0088] like Figure 2 and 3 As shown, the ratio of the total amount of newly generated purified polysaccharides and proteins to the ratio of newly generated purified polysaccharides and proteins using oxygen from H2O is illustrated. Poly and a Prot The values ​​are 6.05 and 6.26 respectively. In the figure, the X-axis represents the newly purified polysaccharides or proteins generated using oxygen from H2O, and the Y-axis represents the total amount of newly purified polysaccharides or proteins generated.

[0089] In this embodiment, based on the average oxygen content percentage of purified polysaccharides and proteins, combined with the total oxygen content of purified polysaccharides and proteins calculated in step S5, and the ratio of the total amount of newly generated purified polysaccharides and proteins to the newly generated portion using H2O, the yield of purified polysaccharides and proteins is accurately calculated using a specific formula. These parameters are interrelated and reflect the utilization of oxygen elements during extracellular polymer synthesis from different perspectives, thereby estimating the actual amount of polysaccharides and proteins produced.

[0090] Specifically, in step S7, the formula for calculating the carbon synthesis rate of the extracellular polymeric polymer is as follows:

[0091]

[0092] Where: 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 soil weighed in step S3, and t is the culture time in step S2.

[0093] The formula for calculating the carbon utilization efficiency of extracellular polymers is as follows:

[0094]

[0095] Among them: CUE EPS C represents the carbon utilization efficiency of extracellular polymers. respiration Represents the carbon flux allocated to respiration (ng C g) −1 h −1 ), C growth Represents the rate of intracellular carbon synthesis by soil microorganisms (ng C g) −1 h −1 );C EPS Represents the rate of extracellular carbon synthesis by soil microorganisms (ng C g) −1 h −1 ).

[0096] In this embodiment, 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, and combined with the polysaccharide and protein yields calculated in step S6, the carbon synthesis rate of extracellular polymers is finally obtained through appropriate mathematical models and calculation methods. The carbon conversion coefficient reflects the conversion relationship between polysaccharides, proteins, and carbon. Using this coefficient, the polysaccharide and protein yields can be converted into the overall carbon synthesis rate of extracellular polymers, intuitively reflecting the efficiency of microbial synthesis of extracellular polymers.

[0097] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A method for analyzing the extracellular polymeric carbon synthesis rate and carbon utilization efficiency, characterized in that, Includes the following steps: S1: Pre-culture the sampled soil; S2: Add an isotope with a purity of 98.0 at%. 18 O-H2O, making the soil 18 A marker group was defined as having an O-H2O content of 20 at%; a group without added isotopes was also included. 18 O-H2O control group; the labeled group and the control group were cultured for 24 hours; S3: The extracellular polymers in the soil of the labeled group and the control group in step S2 were extracted by cation exchange resin method, and purified polysaccharides and proteins were obtained after separation and purification. S4: Collect the gases generated during the cultivation process in step S2 and determine the CO2 concentration using a gas chromatograph; extract soil DNA, determine the DNA concentration, and analyze the DNA sample using an isotope mass spectrometer. 18 O abundance and total oxygen content; obtain soil microbial intracellular carbon synthesis rate and soil microbial respiration rate; The formula for calculating the intracellular carbon synthesis rate of soil microorganisms is as follows: Among them, f DNA It is the ratio of soil microbial biomass carbon content to soil DNA content. produced DW is the yield of double-stranded DNA during incubation, DW is the dry weight of the soil used in step S4, and t is the culture time in step S2. The formula for calculating the soil microbial respiration rate is: in, The value of CO2 generated during the incubation period is given by M, where M is the molecular weight of C, V is the volume of the headspace vial, DW is the dry weight of the soil used in step S2, and t is the incubation time in step S2; 22.4 is the volume of one mole of gas under standard atmospheric pressure. S5: Determination of purified polysaccharide and protein samples using isotope mass spectrometry. 18 The abundance of O and total oxygen content were used to obtain the atomic percentage of purified polysaccharides and proteins, respectively; the calculation process for the atomic percentage of purified polysaccharides and proteins is as follows: ； in: It is atomic percentage over, It refers to the abundance of polysaccharides or proteins in the labeled sample. This refers to the abundance of polysaccharides or proteins in the control group samples; S6: Based on the average oxygen content percentage of the purified polysaccharides and proteins, the total oxygen content of the purified polysaccharides and proteins calculated in step S5, and the ratio of the total amount of newly generated purified polysaccharides and proteins to the newly generated portion of purified polysaccharides and proteins using oxygen from H2O, the yield of purified polysaccharides and proteins is obtained; the average oxygen content percentage of purified polysaccharides and proteins is selected as 29.6% and 38.0%, respectively. The process for obtaining the ratio of the total amount of newly produced purified polysaccharides and proteins to the newly produced portion using oxygen from H2O is as follows: Soil microbial communities containing 18 After culturing the soil extract of O-H2O in LB medium for different time periods, extracellular polymers were extracted using the cation exchange resin method. The changes in the content of purified polysaccharides and proteins in the extracellular polymers before and after culture were analyzed by a chemical colorimetric method, representing the total amount of newly generated purified polysaccharides and proteins. The purified polysaccharides and proteins were then separated and purified, and their content was determined by isotope mass spectrometry. 18 O abundance and total oxygen content, and calculate the newly purified polysaccharides and proteins generated using oxygen from H2O according to the following formula: in: and These represent the polysaccharides and proteins newly generated using oxygen from H2O, respectively. and These are the total oxygen content of the purified polysaccharides and proteins in step S6; It is in the final soil solution of the sample 18 O abundance, which is the abundance of soil in step S2. 18 O-H2O content 20 at%; By fitting a curve between the total amount of newly generated purified polysaccharides and proteins and the newly generated purified polysaccharides and proteins using oxygen from H2O, the ratio of the total amount of newly generated purified polysaccharides and proteins to the newly generated purified polysaccharides and proteins using oxygen from H2O is determined to be a. Poly and a Prot ; S7: Based on the purified polysaccharide, purified protein, carbon conversion coefficient, and the yield of polysaccharide and protein obtained in step S6, obtain the carbon synthesis rate and carbon utilization efficiency of the extracellular polymeric material. The formula for calculating the carbon synthesis rate of extracellular polymers is as follows: Where: 0.40, 0.53 and 0.74 are the carbon conversion coefficients of purified polysaccharide, purified protein and carbon, respectively; DW is the dry weight of soil weighed in step S3 and t is the culture time in step S2; The formula for calculating the carbon utilization efficiency of the extracellular polymeric material is as follows: Among them: CUE EPS C represents the carbon utilization efficiency of extracellular polymers. respiration C represents the soil microbial respiration rate. growth Represents the rate of intracellular carbon synthesis in soil microorganisms; C EPS This represents the rate of extracellular carbon synthesis by soil microorganisms.

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

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

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

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