Methods for promoting grassland methane uptake during the autumn freeze-thaw period through autumn irrigation

Abstract

This invention discloses a method for promoting methane absorption in grasslands during the autumn freeze-thaw period through autumn irrigation. Irrigation is performed on grasslands during the autumn soil moisture sensitive period, defined as a climate-average temperature of 22–10°C. The autumn freeze-thaw period is defined as the start of the period when the minimum soil temperature remains below 0°C for three consecutive days and the end of the period when the maximum soil temperature remains below 0°C for three consecutive days. Three treatments were applied: light rainfall enhancement, moderate rainfall enhancement, and a natural precipitation control. Irrigation was conducted during the autumn soil moisture sensitive period, with a single irrigation volume of 12–15 mm, and 3–4 irrigations were performed throughout the season. The target soil moisture content was maintained at 15%–25%, resulting in a maximum increase of 91.3% in the abundance of the methane oxidation functional gene pmoA. Methane flux increased by 111.7% compared to the control, and methane oxidation potential increased by 97.6%. This method can meet the requirements for grassland greenhouse gas emission reduction under mid-latitude precipitation variation scenarios.

Description

Technical Field

[0001] This invention belongs to the field of environmental protection technology, specifically relating to a method for promoting methane absorption in grasslands during the autumn freeze-thaw period through autumn irrigation. Background Technology

[0002] Methane (CH4) is the world's second largest greenhouse gas, accounting for 16% of total greenhouse gas emissions. As a typical ecosystem in my country's seasonally frozen soil region, the autumn freeze-thaw period (November) is the first peak of methane absorption, and its absorption accounts for more than 30% of the total absorption during the non-growing season.

[0003] Current research on grassland methane uptake largely covers the entire non-growing season, with insufficient attention paid to targeted regulation during the critical autumn freeze-thaw period. Global warming has intensified the freeze-thaw cycle in seasonally frozen soil regions. Changes in autumn precipitation directly affect soil moisture content, porosity, and the activity of methane-oxidizing bacteria, thereby regulating methane uptake during the autumn freeze-thaw period. However, there is a lack of precise gradient schemes for autumn irrigation / water control, and the quantitative impact of increased or decreased water on methane uptake during this period is not clearly defined. Furthermore, the correlation mechanism between autumn water gradients and soil pmoA gene abundance and methane oxidation potential during the autumn freeze-thaw period has not been established, making it impossible to precisely enhance methane uptake during this period through water regulation. Existing technologies have not developed practical schemes adapted to the autumn freeze-thaw period, and data support is limited to the entire non-growing season, lacking quantitative verification of the effects of the autumn freeze-thaw period alone. Summary of the Invention

[0004] To address the aforementioned problems, the present invention provides a method for promoting methane absorption in grasslands during the autumn freeze-thaw period through autumn irrigation.

[0005] A method to promote methane absorption in grasslands during the autumn freeze-thaw period is to irrigate grasslands during the autumn soil moisture-sensitive period.

[0006] The aforementioned sensitive period for soil moisture in autumn is when the average temperature is 22–10℃.

[0007] The autumn freeze-thaw period is defined as follows: the autumn freeze-thaw period begins when the soil temperature remains below 0°C for three consecutive days; and ends when the soil temperature remains below 0°C for three consecutive days.

[0008] The aforementioned autumn freeze-thaw period refers to a soil temperature variation range of -2.3℃ to 5.7℃ for a 5cm soil layer and -1.8℃ to 4.2℃ for a 10cm soil layer.

[0009] The aforementioned sensitive period for soil moisture in autumn is from mid-August to mid-October;

[0010] The irrigation mentioned above is based on natural precipitation, supplementing the total water supply to 150-180 mm;

[0011] The irrigation is carried out in three to four sessions from mid-August to mid-September;

[0012] Each irrigation should maintain a soil moisture content of 15%–25% in the 0-10cm volume.

[0013] The grassland in question belongs to a typical seasonal permafrost distribution area, and the regional climate type is temperate semi-arid continental monsoon climate.

[0014] The present invention has the following beneficial effects:

[0015] 1. Water regulation during key periods focuses on the methane uptake characteristics of Leymus chinensis grasslands during the autumn freeze-thaw period (November). Methane uptake during this period accounts for more than 30% of the total uptake in the non-growing season, with a flux range of 5.25–65.92 μg・m⁻²・h⁻¹. Water regulation is implemented specifically for this period to avoid the indiscriminate application of overall regulation during the non-growing season and to improve the time-specific adaptability of methane uptake control.

[0016] 2. A standardized rainfall enhancement gradient design was implemented, including three treatments: "light rainfall enhancement (W+30%), moderate rainfall enhancement (W+50%), and natural precipitation control." Key technical parameters were clearly defined: irrigation was conducted during the autumn soil moisture-sensitive period (August 14th to October 14th, with an average temperature of 22–10℃), with a single irrigation volume of 12–15 mm, and 3–4 irrigations throughout the season. The target soil moisture content was maintained at 15%–25%, ensuring the reproducibility and practicality of the technology. This resulted in a maximum increase of 91.3% in the abundance of the methane oxidation functional gene pmoA, a 111.7% increase in methane flux compared to the control, and a 97.6% increase in methane oxidation potential.

[0017] 3. Regional Adaptability and Practical Characteristics: This technology is suitable for sheepgrass grasslands in seasonally frozen soil areas such as the Songnen Plain, and is compatible with sandy loam soils and cold temperate climates. No complex equipment is required; it can be implemented through sprinkler irrigation or rain shelters combined with quantitative water replenishment, meeting the practical needs for grassland greenhouse gas emission reduction under mid-latitude precipitation variation scenarios. Attached Figure Description

[0018] Figure 1 The average soil moisture content during the autumn freeze-thaw period under different rainfall increase conditions;

[0019] Figure 2 The abundance of the pmoA gene in soil during the autumn freeze-thaw period under different rainfall increase conditions;

[0020] Figure 3 The amount of methane absorbed during the autumn freeze-thaw period under different rainfall enhancement conditions;

[0021] Figure 4 The amount of methane absorbed during the autumn freeze-thaw period under different rainfall enhancement conditions;

[0022] Figure 5 This is a graph showing the dynamic changes in methane during the autumn freeze-thaw period under different rainfall increase conditions. Detailed Implementation

[0023] Fundamentals of Experimental Design

[0024] 1. Experimental Site: Located in the western core area of ​​the Songnen Plain, a typical area of ​​seasonally frozen soil distribution. The climate type of this region is temperate semi-arid continental monsoon climate, with an average annual temperature of 5.74℃. The non-growing season lasts approximately 220 days, spanning from the end of October to the beginning of April of the following year. The total natural precipitation in autumn (mid-August to mid-October) is approximately 120 mm. The soil type is meadow black calcareous soil, and under natural conditions, the volumetric water content of the 0-10 cm soil layer in autumn ranges from 10% to 15%.

[0025] 2. Criteria for Defining the Autumn Freeze-Thaw Period: Based on the dynamic changes in soil temperature in the study area, the autumn freeze-thaw period was defined using a three-day consecutive soil temperature threshold method: starting from late October, the autumn freeze-thaw period begins when the lowest soil temperature remains below 0℃ for three consecutive days; the autumn freeze-thaw period ends when the highest soil temperature remains below 0℃ for three consecutive days, and the study area enters the stable winter freezing period. The monitoring period for this experiment was from November 2nd to November 20th, 2021. This period met the criteria for defining the autumn freeze-thaw period. During this period, the temperature variation range of the 5cm soil layer was -2.3℃ to 5.7℃, and the temperature variation range of the 10cm soil layer was -1.8℃ to 4.2℃.

[0026] 3. Core Irrigation Principles: Based on the natural precipitation patterns in the study area during autumn, a gradient-based irrigation treatment was implemented. The irrigation period was selected during the autumn soil moisture-sensitive period, when the average temperature ranges from 10℃ to 22℃, corresponding to August 14th to October 14th. The irrigation design focuses on ensuring sufficient water infiltration, aiming to maintain suitable soil moisture content until the autumn freeze-thaw period, while avoiding surface water accumulation that could lead to an anaerobic environment and ensuring the stable progress of the methane oxidation process.

[0027] 4. Monitoring of auxiliary indicators:

[0028] Soil moisture content: The volumetric water content of the 0-10cm soil layer was measured using a TDR300 soil moisture sensor, with a monitoring frequency of once every 3 days. Three replicate measurements were set up for each sampling point, and the final data was the average of the three measurements.

[0029] pmoA gene abundance: Soil samples from the 0–10 cm soil layer were collected at the end of the monitoring period, and the abundance of the methane monooxygenase functional gene (pmoA) was determined using real-time quantitative PCR (q-PCR). The primers used were A189F / Mb661R, with the forward primer (A189F) sequence being GGNGACTGGGACTTCTGG and the reverse primer (Mb661R) sequence being CCGGMGCAACGTCYTTACC. The total volume of the PCR reaction system was 20 μL, specifically comprising: 10 μL SYBR PremixEx Taq II, 0.8 μL each of forward and reverse primers, 2 μL cDNA template, and 6.4 μL ddH2O. The reaction program was set as follows: pre-denaturation at 95℃ for 30s; followed by 40 cycles, each cycle consisting of denaturation at 95℃ for 5s, annealing at 55℃ for 30s, and extension at 72℃ for 30s; finally, the melting curve was analyzed under the conditions of 95℃ for 15s, 60℃ for 1min, and 95℃ for 15s.

[0030] Methane oxidation potential: determined using an indoor isothermal incubation method. 10 g of fresh soil sample was weighed and placed in a 1 L culture bottle. The soil moisture content was adjusted to 34% of field capacity. After sealing, 10 mL of 5% (v / v) CH4 standard gas was injected into the bottle. The bottle was incubated continuously at 25 ℃ in the dark for 5 days. Gas samples were collected at 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h to measure the change in CH4 concentration. The methane oxidation potential was calculated using the following formula:

[0031] P = (ΔC / Δt) × (V / M s ) × (M_CH4 / V m ) × (273 / (273+T))

[0032] In the formula: P is the methane oxidation potential (unit: μg・kg⁻¹・h⁻¹); ΔC / Δt is the rate of change of CH4 concentration in the culture system over time; V is the gas volume in the culture flask (unit: L); M s Mass of soil used for cultivation (unit: kg); M_CH4 is the molar mass of methane; V m T represents the molar volume of gas under standard conditions; T represents the incubation temperature (unit: °C).

[0033] Example 1: Mild Rain Enhancement Irrigation (W+30%)

[0034] 1. Irrigation parameter design

[0035] Increase in water volume: Based on the natural autumn precipitation P=120mm in the study area, an additional 30% precipitation (i.e., 36mm) is added, and the total water supply is 156mm (120mm of natural precipitation + 36mm of irrigation).

[0036] Irrigation frequency and single application amount: Irrigation will be carried out in 3 sessions, 15 days apart (August 14, August 29 and September 13 respectively), with a single irrigation amount of 12 mm (equivalent to 12L of water per square meter).

[0037] Irrigation method: Manual sprinkler irrigation is adopted, with sprinkler head model PY20-40, working pressure 0.2MPa, spray radius 1.5m, ensuring irrigation uniformity ≥85%; irrigation time is selected from 9:00 to 11:00 every day to avoid the impact of high temperature evaporation at noon and low temperature condensation at night on water infiltration.

[0038] Soil moisture content control target: The volumetric moisture content of the 0-10cm soil should be maintained at 15%-20%, which should be monitored in real time using a HOBO small weather station (model UX100-003) at a frequency of once per hour. If the moisture content is lower than 15%, irrigate 5mm.

[0039] 2. Experimental monitoring and data acquisition

[0040] Monitoring period: November 2-20, 2021 (autumn freeze-thaw period), a total of 19 days.

[0041] Methane flux determination (static chamber method) operation procedure:

[0042] 1. Sampling base layout

[0043] In each irrigation treatment plot, three replicate sampling points were set up using random sampling, with the horizontal distance between sampling points controlled at more than 5 m to avoid mutual interference. Seven days before the monitoring work began, a concentric circular iron base (inner diameter 19 cm, outer diameter 23 cm, height 10 cm) was vertically embedded into the soil to a depth of 10 cm, ensuring that the base was in close contact with the surrounding soil without gaps. Distilled water was added to a depth of 2 cm into the annular groove at the top of the base to form a water seal layer to prevent gas exchange between the inside and outside of the chamber during sampling.

[0044] 2. Assembly and commissioning of the gas sampling box

[0045] The gas sampling box is made of cylindrical sheet metal, with dimensions of 20 cm in diameter and 50 cm in height. A small 12 cm × 12 cm DC fan (rated voltage 12 V, rated power 3 W) is embedded in the middle of the side wall of the box to mix the gas inside the box during sampling. The entire exterior of the box is wrapped with aluminum foil to reduce abnormal temperature rise inside the box caused by solar radiation. One sampling port (inner diameter 5 mm) is provided at the top of the box, and the port is equipped with a sealing plug. The plug is replaced with a new one before each sampling to ensure that the port is sealed properly.

[0046] 3. Gas Sample Collection

[0047] Sampling was conducted daily from 9:00 to 11:00. The gas flux monitored during this period showed a 92% agreement with the daily average flux, effectively representing the daily gas exchange level. During sampling, the gas sampling box was precisely attached to the pre-set base, and the connection between the box bottom and the base was immediately sealed with sealing tape. The fan was then activated to mix the air inside the box. Gas was extracted through the sampling port using a 50 mL syringe at four time points: the initial contact time (0 min), 15 min, 30 min, and 45 min. Before each extraction, the syringe was flushed three times with gas from inside the box to remove any residual contamination. After extraction, the gas was quickly injected into a 0.5 L sealed gas bag (model LAE-0.5L). During sampling, the sampling time, the air temperature inside the box, and the soil temperature at a depth of 10 cm were recorded simultaneously. A portable digital thermometer with a measurement accuracy of ±0.1℃ was used for temperature measurement.

[0048] 4. Sample testing and throughput calculation

[0049] The collected gas samples must have their CH4 concentration measured within 7 days. The detection instrument used is a Model 913-1054 methane analyzer manufactured by Los Gatos Research, USA. This instrument has a detection limit of 0.1 ppb and a measurement accuracy of ±1%. The methane flux is calculated based on the slope of the CH4 concentration change over sampling time, using the following formula: F = ρ × h × Δt / Δc × 273.15 + T / 273.15 Where: F is the CH4 flux (unit: μg・m⁻²・h⁻¹); ρ is the density of CH4 under standard conditions (value: 0.717 kg・m⁻³); h is the actual height of the sampling chamber (value: 0.5 m); Δc / Δt is the rate of change of CH4 concentration over time (unit: ppb・h⁻¹); and T is the air temperature inside the chamber during sampling (unit: °C).

[0050] 3. Measurement Results

[0051] Methane flux: The average CH4 uptake during the autumn freeze-thaw period was 41.84±3.26 μg m⁻²h⁻¹, which was significantly higher than that of the natural precipitation control group (P<0.05). The peak occurred on November 12, reaching 52.67 μg m⁻²h⁻¹, which was 79.7% higher than the control.

[0052] Soil moisture content: During the monitoring period, the volumetric moisture content of the soil in the 0-10cm depth ranged from 16.2% to 19.7%, with an average value of 17.9%, which met the preset control target.

[0053] pmoA gene abundance: The pmoA gene abundance in the treatment group was 3.24 × 10⁻⁶. 6 copies g⁻¹, compared to the control group (2.18×10 6 Copies g⁻¹) increased by 48.6%.

[0054] Methane oxidation potential: The methane oxidation potential of the treatment group was 1.87 μg kg⁻¹h⁻¹, which was 52.0% higher than that of the control group (1.23 μg kg⁻¹h⁻¹).

[0055] Example 2: Moderate Rain Enhancement Irrigation W+50%

[0056] 1. Irrigation parameter design

[0057] Increase in water volume: Based on the natural autumn precipitation P=120mm in the study area, an additional 50% precipitation (i.e., 60mm) is added, and the total water supply is 180mm (120mm of natural precipitation + 60mm of irrigation).

[0058] Irrigation frequency and single application amount: Irrigation will be carried out in 4 sessions, with an interval of 12 days (August 14, August 26, September 7, and September 19 respectively), with a single irrigation amount of 15mm (equivalent to 15L of water per square meter).

[0059] Irrigation method: Same as in Example 1, using manual sprinkler irrigation to ensure irrigation uniformity ≥85%; irrigation time is 9:00-11:00 daily.

[0060] Soil moisture content control target: The volumetric moisture content of the 0-10cm soil should be maintained at 20%-25%, which will be monitored in real time using a HOBO mini weather station. If the moisture content is lower than 20%, an additional 8mm of irrigation will be provided.

[0061] 2. Experimental Monitoring and Data Acquisition

[0062] The monitoring period, methane flux determination method (static chamber method), and auxiliary index monitoring (soil moisture content, pmoA gene abundance, methane oxidation potential) were all consistent with those in Example 1, ensuring the uniformity of experimental methods and the comparability of data.

[0063] 3. Measurement Results

[0064] Methane flux: The average CH4 uptake during the autumn freeze-thaw period was 48.35±4.12 μg m⁻²h⁻¹, which was significantly higher than that of the light rain enhancement treatment group and the natural precipitation control group (P<0.05). The peak occurred on November 15, reaching 61.32 μg m⁻²h⁻¹, which was 111.7% higher than the control.

[0065] Soil moisture content: During the monitoring period, the volumetric moisture content of the soil in the 0-10cm depth ranged from 20.5% to 24.3%, with an average value of 22.7%, which met the preset control target.

[0066] pmoA gene abundance: The pmoA gene abundance in the treatment group was 4.17 × 10⁻⁶. 6 The number of copies g⁻¹ increased by 91.3% compared to the control group and by 28.7% compared to the mild rain enhancement treatment group.

[0067] Methane oxidation potential: The methane oxidation potential of the treatment group was 2.43 μg kg⁻¹h⁻¹, which was 97.6% higher than that of the control group and 30.0% higher than that of the mild rain enhancement treatment group.

[0068] Comparative Natural Precipitation Control WCK

[0069] 1. Handling method

[0070] Only natural precipitation (120mm) is accepted, no additional irrigation is required, and other environmental conditions remain the same as in Examples 1 and 2.

[0071] 2. Monitoring and Data Acquisition

[0072] Using the same monitoring methods and frequencies as in Example 1, methane flux was determined using the static chamber method, while soil moisture content, pmoA gene abundance, and methane oxidation potential were monitored simultaneously.

[0073] 3. Measurement Results

[0074] The average CH4 uptake during the autumn freeze-thaw period was 28.97 ± 2.85 μg m⁻²h⁻¹, the average soil volumetric water content in the 0-10 cm depth was 11.3%, and the pmoA gene abundance was 2.18 × 10⁻¹. 6 The methane oxidation potential is 1.23 μg kg⁻¹h⁻¹.

Claims

1. A method for promoting methane absorption in grasslands during the autumn freeze-thaw period through autumn irrigation, characterized by: Irrigate the grassland during the autumn season when soil moisture is sensitive; The aforementioned sensitive period for soil moisture in autumn is when the average temperature is 22–10℃. The autumn freeze-thaw period is defined as follows: the autumn freeze-thaw period begins when the soil temperature is below 0℃ for three consecutive days at its lowest value; and ends when the soil temperature is below 0℃ for three consecutive days at its highest value.

2. The method for promoting methane absorption in grasslands during the autumn freeze-thaw period according to claim 1, characterized in that: The aforementioned autumn freeze-thaw period refers to a soil temperature variation range of -2.3℃ to 5.7℃ for a 5cm soil layer and a soil temperature variation range of -1.8℃ to 4.2℃ for a 10cm soil layer.

3. The method for promoting methane absorption in grasslands during the autumn freeze-thaw period according to claim 3, characterized in that: The aforementioned sensitive period for soil moisture in autumn is from mid-August to mid-October.

4. The method for promoting methane absorption in grasslands during the autumn freeze-thaw period according to claim 1, 2, or 3, characterized in that: The irrigation mentioned above is based on natural precipitation, supplementing the total water supply to 150-180 mm.

5. The method for promoting methane absorption in grasslands during the autumn freeze-thaw period according to claim 4, characterized in that: The irrigation is carried out in three to four sessions from mid-August to mid-September.

6. The method for promoting methane absorption in grasslands during the autumn freeze-thaw period according to claim 5, characterized in that: During each irrigation, the soil moisture content in the 0-10cm volume should be maintained at 15%–25%.

7. The method for promoting methane absorption in grasslands during the autumn freeze-thaw period according to claim 6, characterized in that: The grassland in question belongs to a typical seasonal permafrost distribution area, and the regional climate type is temperate semi-arid continental monsoon climate.

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