Soil organic carbon mineralization rate determination method

Abstract

The invention discloses a soil organic carbon mineralization rate determination method, and relates to the technical field of soil carbon circulation and greenhouse gas monitoring. The method comprises the following steps: carrying out hydrochloric acid back titration on an absorption liquid by adopting an automatic potentiometric titration method, converting to obtain the mass of a carbon element contained in carbon dioxide, calculating the mineralization rate by combining the dry soil weight and the culture interval, and finally subtracting an inactivation control value to obtain the organic carbon mineralization rate driven by pure microorganisms. A thin-wall stainless steel undisturbed soil drill matched with the inner diameter of a culture bottle is used for collecting soil columns in a layered manner, PVC liner tube sealing and constant-temperature transportation are matched, and a cold chamber for simulating the in-situ temperature is combined, so that the in-situ structure and the microbial habitat of the loose high-water-content peat soil are reserved to the maximum extent, and damage of traditional pretreatment to soil pores and microbial communities is avoided.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of soil carbon cycle and greenhouse gas monitoring, and particularly relates to a soil organic carbon mineralization rate determination method. BACKGROUND

[0002] Soil organic carbon mineralization is a key process of releasing carbon dioxide from the soil carbon pool to the atmosphere, and its rate determination is crucial for assessing the carbon balance of an ecosystem. The alkali absorption method is one of the classic soil respiration determination methods, which is based on the principle of using alkaline solution to absorb the carbon dioxide released by soil respiration, and calculating the carbon dioxide release amount by titrating the remaining alkali amount.

[0003] However, the traditional alkali absorption method still has the following problems when applied to plateau peat soil: (1) The plateau peat soil has high water content and loose structure, and the traditional soil sample pretreatment can easily damage the original structure, thereby affecting the microbial activity; (2) The temperature fluctuates greatly in the alpine environment, and the conventional constant temperature culture cannot accurately reflect the temperature sensitivity; (3) The carbon dioxide flux released by peat soil may be low, and the absorption efficiency and determination accuracy of the traditional method are insufficient in long-term culture.

[0004] Therefore, there is an urgent need for an organic carbon mineralization rate determination method that can improve the determination accuracy and environmental simulation authenticity according to the physical and chemical properties of plateau peat soil. SUMMARY

[0005] The purpose of the present application is to provide a soil organic carbon mineralization rate determination method, which solves the above-mentioned many problems of the traditional alkali absorption method applied to plateau peat soil by adopting a stainless steel undisturbed soil drill with a matching culture bottle inner diameter to collect the undisturbed soil column from the peat soil at the preset point, and combining the dry soil weight and the culture interval to calculate the mineralization rate.

[0006] To solve the above technical problems, the present application is realized by the following technical scheme: the present application is a soil organic carbon mineralization rate determination method, comprising the following steps: S01, an undisturbed soil drill (stainless steel undisturbed soil drill) with a size matching the inner diameter of the culture bottle is used to sample the peat soil at the preset point to obtain an undisturbed soil column, which is transported to a cold room for simulation of the in-situ temperature for standing; S02, an undisturbed soil core at a preset depth is placed in a culture bottle, and the dry soil weight of the undisturbed soil core is calculated. An alkali absorption solution mixed with BaCl2 is set in the culture bottle, and a blank control group and an inactivated control group are also set. S03, after sealing the culture bottle, each culture bottle is placed in different gradient temperature simulating natural climate for culture, part of alkali absorption liquid is collected and fresh alkali absorption liquid is injected in the process of culture, and mineralization dynamics is captured; S04, the alkali absorption liquid is titrated by automatic potentiometric titration method, the mass of carbon element contained in carbon dioxide is obtained by conversion, the mineralization rate is calculated combined with the dry soil weight of the original soil core and the culture interval, and finally the pure microbial-driven organic carbon mineralization rate is obtained by subtracting the inactivation control value.

[0007] The application further preferably comprises the following step S01: At a preset peatland collection point, a stainless steel undisturbed soil drill is vertically pressed into the peatland by depth layering, and a complete undisturbed soil column is obtained through a PVC lining pipe in the stainless steel undisturbed soil drill; during the sampling process, the field temperature, moisture condition and vegetation information of each soil layer are recorded throughout the process; The two ends of the PVC lining pipe containing the undisturbed soil column are sealed by using a gas-permeable and pollution-free filter membrane and a sealing cover, and the sealed PVC lining pipe containing the undisturbed soil column is placed in a constant-temperature incubator; After the PVC lining pipe containing the undisturbed soil column is transferred to the cold room, the undisturbed soil column in the PVC lining pipe is pushed out onto a tray containing deionized water-wetted quartz sand, and the cold room is balanced for a preset time period; during the balancing period, the temperature of the cold room is set to be consistent with the annual average ground temperature of the sampling layer.

[0008] The application further preferably comprises the following step S02: A stainless steel cutting ring is used to vertically press into the balanced undisturbed soil column at a specific depth, so as to obtain a cylindrical undisturbed soil core with complete structure, and the wet weight of the undisturbed soil core is weighed; The soil at the same depth adjacent to the undisturbed soil core is dried to a constant weight, the water content of the soil at the depth is determined, and the dry soil weight of the soil sample in the cutting ring is calculated combined with the wet weight and the water content of the undisturbed soil core, wherein the dry soil weight = ; 0.5 mol / L BaCl2 solution is added to each prepared 0.05 mol / L alkali absorption liquid, so that the Ba 2+ The final concentration is 0.02 mol / L, and an absorption cup (optionally a polyethylene centrifuge tube) containing 25.0 mL of alkali absorption liquid mixed with BaCl2 is placed beside the undisturbed soil core in the culture bottle.

[0009] The application further preferably comprises that the blank control group in the step S02 is a culture bottle containing equal amount of alkali absorption liquid (without soil sample), the inactivation control group is a culture bottle containing equal amount of alkali absorption liquid and homologous soil sterilized by 121 DEG C high-pressure steam for 1.0 h and repeatedly sterilized for multiple times, and each culture bottle is cultured synchronously under the same temperature gradient.

[0010] The application further preferably comprises the following step S03: After sealing all the culture bottles, the culture is started, the culture period is 30-60 days, and the culture time points are arranged in the mode of initial densification and later extension, so as to realize complete mineralization dynamic capture through the design of the initial densification and later extension time points; When reaching the sampling time point, a first calibrated airtight syringe is used to puncture the silicone rubber septum on the culture bottle cap to extract the alkali absorption liquid, which is placed in a sealed sample bottle containing BaCl2 solution and marked as to be measured; A second calibrated airtight syringe is used to puncture the silicone rubber septum on the culture bottle cap from a different puncture point, and fresh alkali absorption liquid mixed with BaCl2 is injected into the sampled culture bottle through the second airtight syringe (i.e. the same amount of fresh alkali absorption liquid is injected into the absorption cup in the sampled culture bottle through the second airtight syringe).

[0011] The application further preferably comprises the following step S04: The alkali absorption liquid in the sample bottle to be measured is completely transferred to a 150mL titration cup, 10.0mL of 0.1 mol / L hydrochloric acid standard solution is added to the titration cup, and the excess HCl is back-titrated with 0.05 mol / L NaOH standard solution using an automatic potentiometric titrator, so as to obtain the mass of carbon elements contained in carbon dioxide, calculate the mineralization rate combined with the dry soil weight of the undisturbed soil core and the culture interval, and obtain the pure microbial-driven organic carbon mineralization rate after subtracting the inactivation control value.

[0012] The application further preferably comprises that the mass of CO2 absorbed in the culture bottle containing the undisturbed soil core and the alkali absorption liquid in the step S02 is , the ; wherein the is the volume of NaOH standard solution consumed for titration, the is the volume of NaOH consumed for titration of the same period blank control sample, the is the accurate equivalent concentration of the NaOH standard solution, the value 22.0 is the milliequivalent of CO2, and the is the total volume of the alkali absorption liquid, and the is the volume of the analysis liquid taken out each time.

[0013] The application further preferably comprises that the mass of C elements contained in the CO2 released in the step S04 is calculated by combining the measured mass of CO2 with the molar mass ratio of C to CO2, and the calculation formula is: ; wherein the The molar mass of C is (12.01). The molar mass of CO2 is (44.01); the obtained Combined with the number of days of culture interval dry weight of soil in culture bottle Calculate the average mineralization rate The mineralization rate of the treatment group minus the mineralization rate of the inactivated control group at the same time point yields the organic carbon mineralization rate driven purely by microorganisms. By controlling the reliability of the data through instrument calibration and double parallel samples, the cumulative mineralization amount is obtained by integrating the mineralization rates at each time point. Temperature sensitivity is analyzed using an exponential model, and the main controlling factors of mineralization rate are analyzed in combination with environmental factors.

[0014] The present invention has the following beneficial effects: 1. This invention uses a thin-walled stainless steel undisturbed soil drill with a matching inner diameter of the culture bottle to collect undisturbed soil columns in layers. Combined with PVC liner sealing and constant temperature transportation, and a cold chamber simulating in-situ temperature, this invention preserves the in-situ structure and microbial habitat of loose, high-cement-carbon soil to the maximum extent. This avoids the problem of traditional pretreatment damaging soil pores and microbial communities, thus affecting the accuracy of soil organic carbon mineralization rate measurement results.

[0015] 2. This invention utilizes a dual-syringe non-destructive sampling design to complete the collection of absorbent liquid and replenishment of fresh liquid within seconds, while maintaining the culture bottle sealed throughout the process. This captures the complete dynamics of rapid mineralization in the early stage and slow mineralization in the later stage, while maintaining the stability of the soil sample microenvironment. This makes the indoor monitoring data closer to the actual field conditions and provides a real basis for the accurate assessment of carbon cycling in fragile peatlands. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a flowchart of a method for determining the mineralization rate of soil organic carbon. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1 Please see Figure 1 This invention relates to a method for determining the mineralization rate of soil organic carbon. It demonstrates the complete steps from collecting undisturbed soil columns from peatlands using a stainless steel undisturbed soil drill with a matching inner diameter of the culture bottle, to calculating the mineralization rate by combining dry soil weight and culture interval. Specifically, the method includes the following steps: (1) In-situ soil columns of peatland were collected in layers using a stainless steel undisturbed soil drill with a matching inner diameter to the culture bottle. After being transported under sealed conditions at a constant temperature of 4 ℃, the soil was allowed to stand in a cold room at a simulated in-situ temperature for 48 hours to equilibrate, thus preserving the in-situ soil structure and microbial habitat to the maximum extent. Specifically: Using a specially designed thin-walled, sharp-edged stainless steel burr (with an inner diameter matching the inner diameter of the subsequent 500 mL culture flask, typically 8-10 cm), this burr (i.e., the stainless steel burr) is equipped with a removable, smooth-walled PVC liner.

[0020] At the pre-designated peatland sampling point, the soil drill was vertically pressed into the soil layer by depth (e.g., 0-10 cm, 10-20 cm, 20-40 cm, 40-70 cm, 70-100 cm and below 100 cm), and slowly rotated to obtain an intact original soil column. The two ends of the PVC liner containing the soil column were immediately sealed with a breathable but pollution-proof filter membrane and plastic cap. The sealed soil column was placed vertically in a special 4 ℃ constant temperature insulated box to avoid vibration and transported back to the laboratory within 24 hours. During the sampling process, the field temperature, moisture status and vegetation information of each soil layer were recorded. In a cold chamber at 4±0.5 ℃, carefully push the soil column out of the PVC liner and place it on a tray lined with 2 cm thick quartz sand soaked in deionized water. Allow it to stand in the cold chamber for a preset equilibration time of 48 hours. This process aims to allow the soil pore water to redistribute under gravity, partially restore the soil structure caused by sampling pressure, and stabilize the soil microbial community from transport disturbance. During equilibration, the cold chamber temperature should be set close to the annual average ground temperature of the sampling layer (e.g., 2-4 ℃ for topsoil).

[0021] (2) Obtain undisturbed soil cores at a predetermined depth using a ring cutter and calculate the dry soil weight. Place the cores in a culture flask with a support, and use an optimized NaOH absorbent containing BaCl2. Set up a temperature gradient simulating natural climate and blank and inactivated controls to construct a controllable and reproducible culture system; specifically: Using a stainless steel ring cutter with a sharp inner wall, press the core into the undisturbed soil column at a specific depth, perpendicular to the bedding direction, to obtain a structurally intact cylindrical undisturbed soil core. Quickly weigh the wet weight of the core, and simultaneously take about 10g of soil from an adjacent area at the same depth, dry it at 105 ℃ to constant weight, and determine the moisture content of the soil at that depth. Accurately calculate the dry weight of the soil sample inside the ring cutter using the formula: Soil dry weight = Wet weight of core / (1 + Moisture content), to ensure that the soil quality benchmark is consistent for each culture unit.

[0022] The culture flasks used in the corresponding culture process: use 500 mL wide-mouth glass bottles with screw caps with silicone gaskets to ensure airtightness. Place a porous (pore diameter of about 2 mm) polytetrafluoroethylene (PTFE) or inert plastic support about 3 cm high at the bottom of the bottle.

[0023] The soil sample is placed in the culture bottle by directly and gently transferring the undisturbed soil core from the ring cutter to the support inside the culture bottle, so that the bottom of the soil sample is suspended, which facilitates uniform gas diffusion.

[0024] The temperature gradient setting method during the cultivation process is as follows: the prepared culture bottles are randomly grouped and placed in multiple programmable precision constant temperature incubators. Based on the climate data of the study area, a series of temperature gradients are set, such as: -2 ℃ (simulating freeze-thaw period), 2 ℃ (simulating winter), 6 ℃ (simulating early spring / late autumn), 10 ℃ (simulating early growth season), and 15 ℃ (simulating peak growth season). At least 3 replicates are set for each temperature point.

[0025] The alkaline absorption solution is prepared as follows: Prepare a 0.05 mol / L NaOH absorption solution (compared to the traditional 0.1 mol / L or higher concentrations, this concentration has higher absorption efficiency for low-concentration CO2 and a more acute titration endpoint). Add 0.5 mol / L BaCl2 solution to each portion of the NaOH absorption solution in advance, according to a specified ratio, so that the final BaCl2 concentration is... 2+ With a concentration of approximately 0.02 mol / L, the addition of BaCl2 fixes the absorbed CO2 as BaCO3 precipitate, completely preventing it from being re-evaluated due to pH decrease during long-term incubation or sampling, and significantly improving the accuracy and stability of long-term absorption.

[0026] The absorption cups in the culture flasks are placed as follows: In each culture flask, a 50 mL polyethylene centrifuge tube is placed independently next to the soil sample as an absorption cup. 25.00 mL of the NaOH absorption solution with added BaCl2 is precisely added to the tube. The mouth of the absorption cup should be lower than the bottom of the soil sample to avoid soil splashing in.

[0027] Blank control group (CK): A culture bottle containing only an equal amount of NaOH absorbent solution without soil was set up to correct the background CO2 value in the air that may have seeped into the sealed system during the culture period.

[0028] Inactivated control group (SCK): Culture bottles containing homologous soil that has been autoclaved at 121 °C for 1 hour and repeated 3 times were set up to correct for CO2 that may be generated by abiotic processes in the soil (such as chemical oxidation and mineral decomposition). All control bottles and treatment bottles were cultured synchronously under the same temperature gradient.

[0029] (3) After sealing the culture flask, dynamic culture was carried out for 30 days, with the initial intensification and later extension time points designed. A portion of the absorbent was collected non-destructively using a dual-syringe and fresh absorbent was added immediately to minimize interference while capturing mineralization dynamics; specifically: After sealing all culture flasks, the culture was started with a total culture period of 30 days to fully observe mineralization kinetics. NaOH absorbent was sampled on days 1, 3, 7, 14, 21, and 30 after the start of culture. This design increased the sampling frequency in the early stage when mineralization may be faster, and extended the interval in the later stage to capture complete mineralization dynamics.

[0030] Non-destructive sampling procedure: A puncture-resistant silicone rubber septum is pre-installed on the cap of the culture flask. At the sampling time point, two calibrated airtight syringes are used. The first syringe (10 mL) punctures the septum and accurately extracts 10.00 mL of NaOH absorption solution from the absorption cup. This solution is then quickly injected into a sealed sample bottle that has been pre-filled with 0.5 mL of 0.5 mol / L BaCl2 solution (to ensure complete precipitation of carbonates). The sample bottle is then labeled and ready for testing. Immediately using a second syringe, inject 10.00 mL of freshly prepared 0.05 mol / L NaOH absorption solution containing BaCl2 into the absorption cup through a different puncture point on the same septum to maintain the total volume and absorption capacity of the absorption solution. This operation is completed within seconds, and the culture bottle remains sealed throughout, with the internal O2 concentration, humidity, and pressure environment largely unaffected, achieving true in-situ continuous monitoring.

[0031] (4) The absorbent was back-titrated with hydrochloric acid using an automatic potentiometric titration method to calculate the mass of carbon contained in the carbon dioxide. The mineralization rate was calculated by combining the dry soil weight and the culture interval. Finally, the value of the inactivated control was subtracted to obtain the pure microbial-driven organic carbon mineralization rate; specifically: Transfer all the absorbent from the sample vial to a 150 mL titration vessel. Add 10.00 mL of 0.1 mol / L hydrochloric acid (HCl) standard solution to the titration vessel. The hydrochloric acid will react with all the OH- ions in the absorbent. The reaction of precipitated BaCO3 was investigated using an automatic potentiometric titrator to back-titrate the excess HCl with a 0.05 mol / L NaOH standard solution. The titration endpoint was precisely determined by the instrument via a pH sensor at pH 8.3 (the second theoretical endpoint of carbonic acid, corresponding to the color change point of phenolphthalein indicator), effectively avoiding subjective errors caused by the color of the solution (peat leachate is often colored) in visual titration methods.

[0032] In this embodiment of the invention, the mass of CO2 absorbed in the culture flask containing the undisturbed soil core and NaOH absorption solution is: The ; wherein, the The volume of NaOH standard solution consumed in titrating the sample is... To titrate the volume of NaOH consumed in the concurrent blank control sample, the This refers to the accurate equivalent concentration of the NaOH standard solution, where the value 22.0 represents milligram equivalents of CO2. The total volume of the alkali absorption solution, the This represents the volume of analytical solution extracted each time.

[0033] In this embodiment of the invention, the mass of carbon (C) contained in the CO2 released by mineralization is calculated by combining the measured mass of CO2 with the molar mass ratio of C to CO2, using the following formula: ; Among them, the The molar mass of C is (12.01). The molar mass of CO2 is (44.01); the obtained Combined with the number of days of culture interval dry weight of soil in culture bottle Calculate the average mineralization rate The mineralization rate of the treatment group minus the mineralization rate of the inactivated control group at the same time point yields the organic carbon mineralization rate driven purely by microorganisms. By controlling the reliability of the data through instrument calibration and double parallel samples, the cumulative mineralization amount is obtained by integrating the mineralization rates at each time point. Temperature sensitivity is analyzed using an exponential model, and the main controlling factors of mineralization rate are analyzed in combination with environmental factors.

[0034] Before each titration analysis, the recovery rate of the titration system was tested using a sodium carbonate standard solution of known concentration to ensure measurement accuracy. All sample analyses were performed in duplicate, with a relative deviation requirement of less than 5%. The instantaneous mineralization rate at each time point was plotted as a time-dynamic curve, and the cumulative mineralization over the entire cultivation period was calculated. The average mineralization rate (or cumulative mineralization) at different gradient temperatures was fitted. The commonly used model is the exponential model (…). Model): ,in, The mineralization rate at temperature T. The mineralization rate is used as a reference temperature T0. The value represents the multiple by which the reaction rate increases for every 10 °C increase in temperature. It is a key parameter for assessing the stability of the carbon pool. By combining recorded information on vegetation, water, and other factors, multi-factor statistical analysis is conducted to identify the main controlling factors affecting the mineralization rate.

[0035] Example 2 Please see Figure 1 This invention relates to a method for determining the mineralization rate of soil organic carbon. It demonstrates the complete steps from collecting undisturbed soil columns from peatlands using a stainless steel undisturbed soil drill with a matching inner diameter of the culture bottle, to calculating the mineralization rate by combining dry soil weight and culture interval. Specifically, the method includes the following steps: (1) In-situ soil columns of peatland were collected in layers using a stainless steel undisturbed soil drill with a matching inner diameter to the culture bottle. After being transported under sealed conditions at a constant temperature of 4 ℃, the soil was allowed to stand in a cold room at a simulated in-situ temperature for 48 hours to equilibrate, thus preserving the in-situ soil structure and microbial habitat to the maximum extent. Specifically: Using a specially designed thin-walled, sharp-edged stainless steel burr (with an inner diameter matching the inner diameter of the subsequent 500 mL culture flask, typically 8-10 cm), this burr (i.e., the stainless steel burr) is equipped with a removable, smooth-walled PVC liner.

[0036] At the pre-designated peatland sampling point, the soil drill was vertically pressed into the soil layer by depth (e.g., 0-10 cm, 10-20 cm, 20-40 cm, 40-70 cm, 70-100 cm and below 100 cm), and slowly rotated to obtain an intact original soil column. The two ends of the PVC liner containing the soil column were immediately sealed with a breathable but pollution-proof filter membrane and plastic cap. The sealed soil column was placed vertically in a special 4 ℃ constant temperature insulated box to avoid vibration and transported back to the laboratory within 24 hours. During the sampling process, the field temperature, moisture status and vegetation information of each soil layer were recorded. In a cold chamber at 4±0.5 ℃, carefully push the soil column out of the PVC liner and place it on a tray lined with 2 cm thick quartz sand soaked in deionized water. Allow it to stand in the cold chamber for a preset equilibration time of 48 hours. This process aims to allow the soil pore water to redistribute under gravity, partially restore the soil structure caused by sampling pressure, and stabilize the soil microbial community from transport disturbance. During equilibration, the cold chamber temperature should be set close to the annual average ground temperature of the sampling layer (e.g., 2-4 ℃ for topsoil).

[0037] (2) Obtain undisturbed soil cores at a predetermined depth using a ring cutter and calculate the dry soil weight. Place the cores in a culture flask with a support, and use an optimized KOH absorbent solution containing BaCl2. Set up a temperature gradient simulating natural climate and blank and inactivated controls to construct a controllable and reproducible culture system; specifically: Using a stainless steel ring cutter with a sharp inner wall, press the core into the undisturbed soil column at a specific depth, perpendicular to the bedding direction, to obtain a structurally intact cylindrical undisturbed soil core. Quickly weigh the wet weight of the core, and simultaneously take about 10g of soil from an adjacent area at the same depth, dry it at 105 ℃ to constant weight, and determine the moisture content of the soil at that depth. Accurately calculate the dry weight of the soil sample inside the ring cutter using the formula: Soil dry weight = Wet weight of core / (1 + Moisture content), to ensure that the soil quality benchmark is consistent for each culture unit.

[0038] The culture flasks used in the corresponding culture process: use 500 mL wide-mouth glass bottles with screw caps with silicone gaskets to ensure airtightness. Place a porous (pore diameter of about 2 mm) polytetrafluoroethylene (PTFE) or inert plastic support about 3 cm high at the bottom of the bottle.

[0039] The soil sample is placed in the culture bottle by directly and gently transferring the undisturbed soil core from the ring cutter to the support inside the culture bottle, so that the bottom of the soil sample is suspended, which facilitates uniform gas diffusion.

[0040] The temperature gradient setting method during the cultivation process is as follows: the prepared culture bottles are randomly grouped and placed in multiple programmable precision constant temperature incubators. Based on the climate data of the study area, a series of temperature gradients are set, such as: -2 ℃ (simulating freeze-thaw period), 2 ℃ (simulating winter), 6 ℃ (simulating early spring / late autumn), 10 ℃ (simulating early growth season), and 15 ℃ (simulating peak growth season). At least 3 replicates are set for each temperature point.

[0041] The alkaline absorption solution is prepared as follows: Prepare a 0.05 mol / L KOH absorption solution (compared to the traditional 0.1 mol / L or higher concentrations, this concentration has higher absorption efficiency for low-concentration CO2 and a more acute titration endpoint). Add 0.5 mol / L BaCl2 solution to each portion of KOH absorption solution in advance, according to a specified ratio, so that the final BaCl2 concentration is... 2+ With a concentration of approximately 0.02 mol / L, the addition of BaCl2 fixes the absorbed CO2 as BaCO3 precipitate, completely preventing it from being re-evaluated due to pH decrease during long-term incubation or sampling, and significantly improving the accuracy and stability of long-term absorption.

[0042] The absorption cups in the culture flasks are placed as follows: In each culture flask, a 50 mL polyethylene centrifuge tube is placed independently next to the soil sample as an absorption cup. 25.00 mL of the KOH absorption solution with added BaCl2 is precisely added to the tube. The mouth of the absorption cup should be lower than the bottom of the soil sample to avoid soil splashing in.

[0043] Blank control group (CK): A culture bottle containing only an equal amount of KOH absorbent solution without soil was set up to correct the background CO2 value in the air that may have seeped into the sealed system during the culture period.

[0044] Inactivated control group (SCK): Culture bottles containing homologous soil that has been autoclaved at 121 °C for 1 hour and repeated 3 times were set up to correct for CO2 that may be generated by abiotic processes in the soil (such as chemical oxidation and mineral decomposition). All control bottles and treatment bottles were cultured synchronously under the same temperature gradient.

[0045] (3) After sealing the culture flask, dynamic culture was carried out for 45 days, with the initial intensification and later extension time points designed. A portion of the absorbent was collected non-destructively using a dual-syringe and fresh absorbent was added immediately to minimize interference while capturing mineralization dynamics; specifically: After sealing all culture flasks, the culture was started with a total culture period of 45 days to fully observe mineralization kinetics. KOH absorbent was sampled on days 1, 3, 7, 14, 21, 30, and 45 after the start of culture. This design increased the sampling frequency in the early stage when mineralization may be faster and extended the interval in the later stage to capture complete mineralization dynamics.

[0046] Non-destructive sampling procedure: A puncture-resistant silicone rubber septum is pre-installed on the cap of the culture flask. At the sampling time point, two calibrated airtight syringes are used. The first syringe (10 mL) punctures the septum and accurately extracts 10.00 mL of KOH absorption solution from the absorption cup. This solution is then quickly injected into a sealed sample bottle that has been pre-filled with 0.5 mL of 0.5 mol / L BaCl2 solution (to ensure complete precipitation of carbonates). The sample bottle is then labeled and ready for testing. Immediately using a second syringe, inject 10.00 mL of freshly prepared 0.05 mol / L KOH absorption solution containing BaCl2 into the absorption cup through different puncture points on the same septum to maintain the total volume and absorption capacity of the absorption solution. This operation is completed within seconds, and the culture bottle remains sealed throughout, with the internal O2 concentration, humidity, and pressure environment largely unaffected, achieving true in-situ continuous monitoring.

[0047] (4) The absorbent was back-titrated with hydrochloric acid using an automatic potentiometric titration method to calculate the mass of carbon contained in the carbon dioxide. The mineralization rate was calculated by combining the dry soil weight and the culture interval. Finally, the value of the inactivated control was subtracted to obtain the pure microbial-driven organic carbon mineralization rate; specifically: Transfer all the absorbent from the sample vial to a 150 mL titration vessel. Add 10.00 mL of 0.1 mol / L hydrochloric acid (HCl) standard solution to the titration vessel. The hydrochloric acid will react with all the OH- ions in the absorbent. The reaction of precipitated BaCO3 was investigated using an automatic potentiometric titrator to back-titrate the excess HCl with a 0.05 mol / L NaOH standard solution. The titration endpoint was precisely determined by the instrument via a pH sensor at pH 8.3 (the second theoretical endpoint of carbonic acid, corresponding to the color change point of phenolphthalein indicator), effectively avoiding subjective errors caused by the color of the solution (peat leachate is often colored) in visual titration methods.

[0048] Example 3 Please see Figure 1 This invention relates to a method for determining the mineralization rate of soil organic carbon. It demonstrates the complete steps from collecting undisturbed soil columns from peatlands using a stainless steel undisturbed soil drill with a matching inner diameter of the culture bottle, to calculating the mineralization rate by combining dry soil weight and culture interval. Specifically, the method includes the following steps: (1) In-situ soil columns of peatland were collected in layers using a stainless steel undisturbed soil drill with a matching inner diameter to the culture bottle. After being transported under sealed conditions at a constant temperature of 4 ℃, the soil was allowed to stand in a cold room at a simulated in-situ temperature for 48 hours to equilibrate, thus preserving the in-situ soil structure and microbial habitat to the maximum extent. Specifically: Using a specially designed thin-walled, sharp-edged stainless steel burr (with an inner diameter matching the inner diameter of the subsequent 500 mL culture flask, typically 8-10 cm), this burr (i.e., the stainless steel burr) is equipped with a removable, smooth-walled PVC liner.

[0049] At the pre-designated peatland sampling point, the soil drill was vertically pressed into the soil layer by depth (e.g., 0-10 cm, 10-20 cm, 20-40 cm, 40-70 cm, 70-100 cm and below 100 cm), and slowly rotated to obtain an intact original soil column. The two ends of the PVC liner containing the soil column were immediately sealed with a breathable but pollution-proof filter membrane and plastic cap. The sealed soil column was placed vertically in a special 4 ℃ constant temperature insulated box to avoid vibration and transported back to the laboratory within 24 hours. During the sampling process, the field temperature, moisture status and vegetation information of each soil layer were recorded. In a cold chamber at 4±0.5 ℃, carefully push the soil column out of the PVC liner and place it on a tray lined with 2 cm thick quartz sand soaked in deionized water. Allow it to stand in the cold chamber for a preset equilibration time of 48 hours. This process aims to allow the soil pore water to redistribute under gravity, partially restore the soil structure caused by sampling pressure, and stabilize the soil microbial community from transport disturbance. During equilibration, the cold chamber temperature should be set close to the annual average ground temperature of the sampling layer (e.g., 2-4 ℃ for topsoil).

[0050] (2) Obtain undisturbed soil cores at a predetermined depth using a ring cutter and calculate the dry soil weight. Place the cores in a culture flask with a support, and use an optimized NaOH absorbent containing BaCl2. Set up a temperature gradient simulating natural climate and blank and inactivated controls to construct a controllable and reproducible culture system; specifically: Using a stainless steel ring cutter with a sharp inner wall, press the core into the undisturbed soil column at a specific depth, perpendicular to the bedding direction, to obtain a structurally intact cylindrical undisturbed soil core. Quickly weigh the wet weight of the core, and simultaneously take about 10g of soil from an adjacent area at the same depth, dry it at 105 ℃ to constant weight, and determine the moisture content of the soil at that depth. Accurately calculate the dry weight of the soil sample inside the ring cutter using the formula: Soil dry weight = Wet weight of core / (1 + Moisture content), to ensure that the soil quality benchmark is consistent for each culture unit.

[0051] The culture flasks used in the corresponding culture process: use 500 mL wide-mouth glass bottles with screw caps with silicone gaskets to ensure airtightness. Place a porous (pore diameter of about 2 mm) polytetrafluoroethylene (PTFE) or inert plastic support about 3 cm high at the bottom of the bottle.

[0052] The soil sample is placed in the culture bottle by directly and gently transferring the undisturbed soil core from the ring cutter to the support inside the culture bottle, so that the bottom of the soil sample is suspended, which facilitates uniform gas diffusion.

[0053] The temperature gradient setting method during the cultivation process is as follows: the prepared culture bottles are randomly grouped and placed in multiple programmable precision constant temperature incubators. Based on the climate data of the study area, a series of temperature gradients are set, such as: -2 ℃ (simulating freeze-thaw period), 2 ℃ (simulating winter), 6 ℃ (simulating early spring / late autumn), 10 ℃ (simulating early growth season), and 15 ℃ (simulating peak growth season). At least 3 replicates are set for each temperature point.

[0054] The alkaline absorption solution is prepared as follows: Prepare a 0.05 mol / L NaOH absorption solution (compared to the traditional 0.1 mol / L or higher concentrations, this concentration has higher absorption efficiency for low-concentration CO2 and a more acute titration endpoint). Add 0.5 mol / L BaCl2 solution to each portion of the NaOH absorption solution in advance, according to a specified ratio, so that the final BaCl2 concentration is...2+ With a concentration of approximately 0.02 mol / L, the addition of BaCl2 fixes the absorbed CO2 as BaCO3 precipitate, completely preventing it from being re-evaluated due to pH decrease during long-term incubation or sampling, and significantly improving the accuracy and stability of long-term absorption.

[0055] The absorption cups in the culture flasks are placed as follows: In each culture flask, a 50 mL polyethylene centrifuge tube is placed independently next to the soil sample as an absorption cup. 25.00 mL of the NaOH absorption solution with added BaCl2 is precisely added to the tube. The mouth of the absorption cup should be lower than the bottom of the soil sample to avoid soil splashing in.

[0056] Blank control group (CK): A culture bottle containing only an equal amount of NaOH absorbent solution without soil was set up to correct the background CO2 value in the air that may have seeped into the sealed system during the culture period.

[0057] Inactivated control group (SCK): Culture bottles containing homologous soil that has been autoclaved at 121 °C for 1 hour and repeated 3 times were set up to correct for CO2 that may be generated by abiotic processes in the soil (such as chemical oxidation and mineral decomposition). All control bottles and treatment bottles were cultured synchronously under the same temperature gradient.

[0058] (3) After sealing the culture flask, dynamic culture was carried out for 60 days, with the initial intensification and later extension time points designed. A portion of the absorbent was collected non-destructively using a dual-syringe and fresh absorbent was added immediately to minimize interference while capturing mineralization dynamics; specifically: After sealing all culture flasks, the culture was started with a total culture period of 60 days to fully observe mineralization kinetics. NaOH absorbent was sampled on days 1, 3, 7, 14, 21, 30, 45, and 60 after the start of culture. This design increased the sampling frequency in the early stage when mineralization may be faster, and extended the interval in the later stage to capture complete mineralization dynamics.

[0059] Non-destructive sampling procedure: A puncture-resistant silicone rubber septum is pre-installed on the cap of the culture flask. At the sampling time point, two calibrated airtight syringes are used. The first syringe (10 mL) punctures the septum and accurately extracts 10.00 mL of NaOH absorption solution from the absorption cup. This solution is then quickly injected into a sealed sample bottle that has been pre-filled with 0.5 mL of 0.5 mol / L BaCl2 solution (to ensure complete precipitation of carbonates). The sample bottle is then labeled and ready for testing. Immediately using a second syringe, inject 10.00 mL of freshly prepared 0.05 mol / L NaOH absorption solution containing BaCl2 into the absorption cup through a different puncture point on the same septum to maintain the total volume and absorption capacity of the absorption solution. This operation is completed within seconds, and the culture bottle remains sealed throughout, with the internal O2 concentration, humidity, and pressure environment largely unaffected, achieving true in-situ continuous monitoring.

[0060] (4) The absorbent was back-titrated with hydrochloric acid using an automatic potentiometric titration method to calculate the mass of carbon contained in the carbon dioxide. The mineralization rate was calculated by combining the dry soil weight and the culture interval. Finally, the value of the inactivated control was subtracted to obtain the pure microbial-driven organic carbon mineralization rate; specifically: Transfer all the absorbent from the sample vial to a 150 mL titration vessel. Add 10.00 mL of 0.1 mol / L hydrochloric acid (HCl) standard solution to the titration vessel. The hydrochloric acid will react with all the OH- ions in the absorbent. The reaction of precipitated BaCO3 was investigated using an automatic potentiometric titrator to back-titrate the excess HCl with a 0.05 mol / L NaOH standard solution. The titration endpoint was precisely determined by the instrument via a pH sensor at pH 8.3 (the second theoretical endpoint of carbonic acid, corresponding to the color change point of phenolphthalein indicator), effectively avoiding subjective errors caused by the color of the solution (peat leachate is often colored) in visual titration methods.

[0061] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0062] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for measuring the rate of mineralization of soil organic carbon, characterized by, The method comprises the following steps: S01, using undisturbed soil drill matched with the inner diameter size of the culture bottle, layered sampling of the peatland at the preset point to obtain an undisturbed soil column, and transporting the undisturbed soil column to a cold room for simulation of in-situ temperature for standing; S02, obtaining an undisturbed soil core at a preset depth and placing the undisturbed soil core in a culture bottle, and calculating the dry soil weight of the undisturbed soil core, setting alkaline absorption liquid mixed with BaCl2 in the culture bottle and separate from the undisturbed soil core, and setting a blank control group and an inactivated control group; S03, after sealing the culture bottle, placing each culture bottle in different gradient temperatures simulating natural climate for culture, collecting part of the alkaline absorption liquid in a sealed manner and injecting fresh alkaline absorption liquid in the culture process, and capturing the mineralization dynamics; S04, using automatic potentiometric titration method to perform hydrochloric acid back titration on the alkaline absorption liquid, converting to obtain the carbon element mass contained in carbon dioxide, and calculating the mineralization rate combined with the dry soil weight of the undisturbed soil core and the culture interval, and obtaining the pure microbial-driven organic carbon mineralization rate after subtracting the inactivated control value.

2. The method of claim 1, wherein the soil organic carbon mineralization rate is determined by the following equation: ###0001### wherein, R is the soil organic carbon mineralization rate, C is the soil organic carbon concentration, t is the time, and k is the soil organic carbon mineralization rate constant. The step S01 specifically comprises: At the preset peatland collection point, a stainless steel undisturbed soil drill is vertically pressed into the peatland according to the depth, a complete undisturbed soil column is obtained by rotating the stainless steel undisturbed soil drill through the PVC lining pipe in the stainless steel undisturbed soil drill, and the field temperature, moisture condition and vegetation information of each soil layer are recorded during the sampling process; A gas-permeable and pollution-free filter membrane and a sealing cover are used to seal the two ends of the PVC lining pipe containing the undisturbed soil column, and the sealed PVC lining pipe containing the undisturbed soil column is placed in a constant temperature incubator; After the PVC lining pipe containing the undisturbed soil column is transferred to the cold room, the undisturbed soil column in the PVC lining pipe is pushed out to a tray containing deionized water wetted quartz sand, and the cold room is balanced for a preset time, and the temperature of the cold room is set to be consistent with the annual average ground temperature of the sampling horizon during the balancing period.

3. The method of claim 1, wherein the soil organic carbon mineralization rate is determined by the following equation: ###0001### wherein, R is the soil organic carbon mineralization rate, C is the soil organic carbon concentration, t is the time, and k is the soil organic carbon mineralization rate constant. The step S02 specifically comprises: A stainless steel ring knife is used to vertically press into the specific depth of the balanced undisturbed soil column perpendicular to the bedding direction to obtain a cylindrical undisturbed soil core with complete structure, and the wet weight of the undisturbed soil core is weighed; The soil adjacent to the same depth is dried to constant weight, the moisture content of the soil at the depth is determined, and the dry soil weight of the soil sample in the cutting ring is calculated based on the wet weight and the moisture content of the undisturbed soil core, wherein the dry soil weight = the wet weight of the undisturbed soil core x (100% - the moisture content of the undisturbed soil core) / 100% ; In each of the prepared 0.05 mol / L alkali absorption solution, 0.5 mol / L BaCl2 solution was added to make the Ba 2+ The final concentration was 0.02 mol / L, and the absorption cup containing 25.0 mL of alkali absorption solution mixed with BaCl2 was placed beside the undisturbed soil core in the culture bottle.

4. The method of claim 1, wherein the soil organic carbon mineralization rate is determined by the following equation: ###0001### wherein, R is the soil organic carbon mineralization rate, C is the soil organic carbon concentration, t is the time, and k is the soil organic carbon mineralization rate constant. The blank control group in the step S02 is a culture bottle containing an equal amount of alkaline absorption liquid, and the inactivated control group is a culture bottle containing an equal amount of alkaline absorption liquid and the same source soil sterilized by 121 DEG C high-pressure steam for 1.0 h and repeatedly sterilized for multiple times, and each culture bottle is cultured synchronously under the same temperature gradient.

5. The method for determining the soil organic carbon mineralization rate according to claim 1, characterized in that, The step S03 specifically comprises: After all the culture bottles are sealed, the culture is started, the culture period is 30-60 days, and the culture time points are arranged in the manner of initial intensive and later extension, and the complete mineralization dynamic is captured through the design of the initial intensive and later extension time points; When the sampling time point is reached, a first calibrated airtight syringe is used to pierce the silicone rubber septum on the culture bottle cap to extract the alkaline absorption liquid, which is placed in a sealed sample bottle containing BaCl2 solution and marked for testing; A second calibrated airtight syringe is used to pierce the silicone rubber septum on the culture bottle cap from different piercing points, and fresh alkaline absorption liquid mixed with BaCl2 is injected into the sampled culture bottle through the second airtight syringe.

6. The method of claim 1, wherein the soil organic carbon mineralization rate is determined by the method comprising: The step S04 specifically comprises: ​ The alkali absorption liquid in the sample bottle to be tested is transferred to a 150 mL titration cup, 10.0 mL of a 0.1 mol / L hydrochloric acid standard solution is added to the titration cup, and the excess HCl is back titrated with a 0.05 mol / L NaOH standard solution using an automatic potentiometric titrator. The mass of carbon contained in the carbon dioxide is converted to obtain the mineralization rate in combination with the dry soil weight of the undisturbed soil core and the incubation interval. After subtracting the inactivation control value, the pure microbial-driven organic carbon mineralization rate is obtained.

7. The method of claim 1, wherein the method is characterized by, The mass of CO2 absorbed in the culture bottle equipped with the undisturbed soil core and the alkali absorption solution in the step S02 is , and the ; wherein the is the volume of NaOH standard consumed for titrating the sample, the is the volume of NaOH consumed for titrating the blank sample, the is the accurate equivalent concentration of the NaOH standard solution, the value 22.0 is the milliequivalent of CO2, the is the total volume of the base absorption solution, the is the volume of the analysis solution taken out each time.

8. The method of claim 1, wherein the soil organic carbon mineralization rate is determined by the method comprising: The mass of C element contained in the CO2 released in the step S04 is calculated by combining the measured CO2 mass with the molar mass ratio of C to CO2, and the calculation formula is: ​ 9. wherein, said molar mass of C, said molar mass of CO2; The obtained In combination with the culture interval days With the soil dry weight in the culture bottle , the average mineralization rate was calculated .

Images