Soil culture method and system for simulating natural ecological conditions

Abstract

The invention provides a soil culture system capable of simulating natural ecological conditions. The soil culture system comprises an undisturbed soil structure maintaining structure, and a soil-water-gas interface regulation and control structure is arranged in the undisturbed soil structure maintaining structure; the undisturbed soil structure maintaining structure comprises a culture bottle; the soil-water-gas interface regulation and control structure comprises a soil simulation culture unit, and a water-gas balance nested base is arranged at the lower end of the soil simulation culture unit. The invention also provides a soil culture method for simulating natural ecological conditions, which comprises the following steps: filling the soil into the soil simulation culture unit, then mounting the water-gas balance nested base at the lower end of the soil simulation culture unit filled with the soil, then putting the soil simulation culture unit into the culture bottle, and then adding ultrapure water into the bottom of the culture bottle. According to the system, the natural state is simulated by controlling the soil bulk density, water and air permeability of the bottom and capillary water replenishing, and an approximately natural habitat can be created for soil carbon and nitrogen element turnover and soil microorganism growth, so that the accuracy of isotope labeling research is greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of soil cultivation technology, specifically relating to a soil cultivation method and system that simulates natural ecological conditions. Background Technology

[0002] Soil culture systems are crucial platforms for isotope labeling experiments, and their design directly impacts the reliability of experimental data and the accuracy of ecological interpretation. An ideal culture device should be able to simulate natural or near-natural soil environments as realistically as possible under controlled conditions, including maintaining key parameters such as bulk density, moisture dynamics, aeration conditions, and temperature fluctuations similar to in-situ soil. However, existing soil culture devices struggle to achieve a balance between precise control of experimental conditions and simulation of natural ecological states, and a satisfactory solution has yet to be found.

[0003] Traditional soil culture devices utilize closed sampling systems and ventilated, negative-pressure-proof designs, but do not isolate the reaction chamber from the sample chamber. Instead, they directly fill glass bottles with loose soil to achieve atmospheric pressure closed culture and in-situ gas collection, creating a microenvironment conducive to isotope determination. These methods have been widely used in laboratory studies of organic matter mineralization, evoked effects, and greenhouse gas production, and related research is frequently reported. However, improvements to traditional culture devices can only add functionality and perform localized optimizations within existing closed containers. They cannot effectively simulate the physical structure and water-air dynamic balance of soil under natural conditions, leading to fundamental problems such as uncontrolled soil bulk density, the formation of anaerobic environments at the bottom, insufficient purging of synthetic air, and reliance on manual intervention for water management. This, in turn, increases the discrepancy between experimental data and real soil ecological processes.

[0004] Traditional, simple glass bottle culture systems have serious shortcomings, making it difficult to simulate real soil environments. Specifically, due to the sealed bottom of the container, soil pore water cannot drain properly, resulting in an anaerobic soil system, which does not match the aerobic conditions of most terrestrial ecosystems. Because of the sealed bottom, if ventilation is not provided for more than 48 hours, noticeable mold will appear at the bottom of the soil. The complete contact between the soil and the bottom of the traditional culture device prevents comprehensive artificial synthetic air purging and carbon dioxide extraction during cultivation. Furthermore, the simple soil filling process completely ignores the crucial physical parameter of soil bulk density, leading to a significant difference between the soil structure and its natural state. In addition, frequent artificial watering not only introduces interference but also causes continuous fluctuations in soil moisture content, making it impossible to maintain the steady-state conditions required for the experiment, ultimately significantly affecting the accuracy and reliability of the experimental data.

[0005] Against this backdrop, it is of great significance to develop a system that simulates the natural state by controlling soil bulk density, bottom permeability and aeration, and capillary water replenishment. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a soil cultivation method and system that simulates natural ecological conditions to address the shortcomings of the prior art. The soil cultivation system of the present invention simulates the natural state by controlling the soil bulk density, bottom water permeability and aeration and realizing capillary water replenishment. It can create a near-natural growth environment for soil microorganisms to participate in the soil carbon and nitrogen element turnover process, so as to greatly improve the accuracy of isotope labeling research.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a soil cultivation system that simulates natural ecological conditions, the soil cultivation system including an undisturbed soil structure maintenance structure, and a soil-water-air interface regulation structure is provided within the undisturbed soil structure maintenance structure; The original soil structure maintenance structure includes a culture bottle, the upper end of which is provided with a bottle cap, and an air passage pipe is provided through the center of the bottle cap. A multi-channel air passage control valve is provided on the air passage pipe. The soil-water-air interface control structure includes a soil simulation culture unit, a water-air balance nested base is provided at the lower end of the soil simulation culture unit, multiple holes are provided on the water-air balance nested base, multiple rows of holes are provided on the side wall of the water-air balance nested base, and a water-permeable and air-permeable support membrane is wrapped at the lower end of the water-air balance nested base.

[0008] Preferably, the soil simulation culture unit is a PVC ring cutter, the inner diameter of which is 46 mm, the height is 60 mm, and the volume is 100 cm³. 3 .

[0009] Preferably, the water-permeable and air-permeable support membrane is made of nylon fabric with a pore size of 400 mesh; the water-air balance nested base is made of PVC.

[0010] Preferably, the water-air balance nested base has 69 openings, and the side wall of the water-air balance nested base has 4 rows of openings, with 3 openings in each row; the openings are circular, and the radius of the circle is 1 mm.

[0011] This invention also provides a soil cultivation method simulating natural ecological conditions, the method comprising the following steps: The soil was filled to a height calculated based on the natural soil bulk density. Based on this filling height, the soil was divided into four equal layers and filled into the soil simulation culture unit. Each layer of soil was compacted and cleaned before the next layer was filled. Stable isotope markers were then added to the surface of the soil simulation culture unit. The water-air balance nested base was then installed at the bottom of the soil simulation culture unit filled with soil, and then it was placed in the culture bottle. Ultrapure water was added to the bottom of the culture bottle, and ultrapure water was added along the inner wall of the culture bottle every 20 days.

[0012] Preferably, the amount of soil used is 100 g; the compaction is performed using a soil compactor, the cleaning is performed using a PVC brush, and the cleaning time is 30 s.

[0013] Preferably, 2 mg of the stable isotope marker is added per gram of soil; 5 mL of ultrapure water is added to the bottom of the culture flask, and 5 mL of ultrapure water is added along the inner wall of the culture flask every 20 days.

[0014] Compared with the prior art, the present invention has the following advantages: 1. The present invention aims to provide a method and apparatus that can accurately control and simulate the bulk density of natural soil, while creating a physical and aeration environment for the soil that is consistent with its natural state through a bottom permeable and aeration design.

[0015] 2. This invention provides an automatic water replenishment system based on the principle of capillary action, which can continuously and stably maintain soil moisture without damaging the soil structure, thereby effectively solving the problems of interference and humidity fluctuation caused by frequent manual water replenishment in traditional methods.

[0016] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the soil cultivation system for simulating natural ecological conditions according to the present invention; Figure 2 The effect of different incubation devices on soil bulk density is indicated by different lowercase letters, which indicate significant differences between treatments at the p<0.05 level. Figure 3 The effect of different culture devices on soil porosity is indicated by different lowercase letters, which indicate significant differences between treatments at the p<0.05 level. Figure 4 This represents the effect of different culture devices on soil microbial biomass carbon content. Different lowercase letters indicate significant differences between treatments at the p<0.05 level; MBC represents microbial biomass carbon. Figure 5This represents the effect of different culture devices on soil β-glucosidase content. Different lowercase letters indicate significant differences between treatments at the p<0.05 level; BG represents β-glucosidase. Figure 6 The effect of different culture devices on soil microbial carbon use efficiency is shown. Different lowercase letters indicate significant differences between treatments at the p<0.05 level; CUE represents microbial carbon use efficiency. Figure 7 In different culture devices 13 Dynamics of cumulative CO2 release; Figure 8 It is a traditional soil cultivation device; Figure 9 It is a diagram recording the prototype design and testing process; Figure 10 This is a physical image of the soil cultivation system that simulates natural ecological conditions according to the present invention; Figure 11 It is a photo of the original soil in which the soil was cultivated.

[0018] Explanation of reference numerals in the attached diagram: 1. Original soil structure maintenance structure; 101. Culture bottle; 102. Multi-channel gas control valve; 2. Soil-water-air interface regulation structure; 201. Soil simulation culture unit; 202. Water-air balance nested base; 203. Water-permeable and air-permeable support membrane. Detailed Implementation

[0019] 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. Example 1

[0020] This embodiment provides a soil cultivation system that simulates natural ecological conditions. The soil cultivation system includes an undisturbed soil structure maintenance structure 1, and a soil-water-air interface regulation structure 2 is provided inside the undisturbed soil structure maintenance structure 1. The original soil structure maintenance structure 1 includes a culture bottle 101, with a bottle cap at the upper end of the culture bottle 101. An air passage pipe is provided through the center of the bottle cap, and a multi-channel air passage control valve 102 is provided on the air passage pipe. The soil-water-air interface regulation structure 2 includes a soil simulation culture unit 201. A water-air balance nested base 202 is installed at the lower end of the soil simulation culture unit 201. The water-air balance nested base 202 has 69 circular openings running through it, and four rows of three circular openings in each row, totaling 12 openings, on its sidewalls. All circular openings have a radius of 1 mm, designed based on soil aeration and rapid water transport characteristics to ensure gas exchange and water-air balance at the bottom of the soil column. The lower end of the water-air balance nested base 202 is wrapped with a water-permeable and air-permeable support membrane 203, which is made of nylon cloth with a pore size of 400 mesh. Because medical sterile gauze absorbs water too quickly and is expensive, and phosphate-free filter paper has problems such as rapid water absorption, unstable water absorption, and fragility, nylon cloth with stable water absorption and low cost is chosen. The water-air balance nested base 202 is made of PVC, and the pore size radius is set to 1 mm based on soil aeration and rapid water transport characteristics, compared to a gridded pore structure (…). Figure 9 It is more in line with the natural state and has a lower production cost; Soil simulation culture unit 201 has an inner diameter of 46 mm, a height of 60 mm, and a volume of 100 cm³. 3 The PVC ring cutter; its core function is not simply "filling with soil", but rather to act as a geometric constraint boundary to ensure that the soil can be precisely filled and fixed according to the natural bulk density, thereby completely preserving and reproducing the soil's pore structure, aggregate state, and microbial habitat in the field in the laboratory. This is the physical basis for obtaining ecological authenticity; the size of the soil simulation culture unit 201 is determined based on the average soil usage of 100 g in the culture experiment; ensuring that the undisturbed soil can be directly used for X-ray micro-CT scanning analysis of soil porosity and other indicators after the culture is completed; The water-air balance nested base 202 is not a simple filter or isolation component; its core function is to simulate the interface function between different soil layers in natural soil. It can act as a "biomimetic capillary bundle": through the capillary force generated by its microporous fiber structure, it actively and continuously transports free water from the bottom to the soil layer, realizing automatic, stable, and undisturbed replenishment of soil moisture, completely replacing the traditional manual tubular water replenishment method. The nylon cloth acts as a "selective permeable membrane": it allows gases such as oxygen and carbon dioxide to pass through freely, ensuring that the soil system is always in an aerobic environment consistent with nature, while effectively preventing the loss of soil particles. The multi-channel gas path control valve 102 (three-way valve) upgrades the traditional culture device into a controllable dynamic gas path system. It enables free switching between three modes—"closed culture," "synthetic air injection," and "isotope-labeled gas sampling and analysis"—without disrupting the culture microenvironment, providing key technical support for in-situ, quantitative, and dynamic monitoring of the carbon and nitrogen turnover process. Example 2

[0021] An example provides a soil cultivation method that simulates natural ecological conditions, the method comprising the following steps: 100 g of soil was used to calculate the filling height based on the bulk density of the natural soil collected in the experiment. Then, based on the calculated filling height, it was divided into four layers and filled into the soil simulation culture unit 201. Each layer of soil was compacted using a soil compactor and cleaned with a PVC brush for 30 seconds before filling the next layer to ensure that the soil layers were uniform and in good contact. Stable isotope markers were added to the surface of the soil simulation culture unit 201 using a pipette. The amount of stable isotope-labeled carbon added per gram of soil was 2 mg to induce microbial growth. Then, a water-air balance nested base 202 was installed at the bottom of the soil simulation culture unit 201 filled with soil. After that, it was placed in the culture bottle 101. 5 mL of ultrapure water was added to the bottom of the culture bottle 101. Every 20 days, 5 mL of ultrapure water was added along the inner wall of the culture bottle 101 using a pipette to ensure that the nylon cloth in the water-air balance nested base 202 continuously and stably replenished the soil when it needed water. The formula for calculating the filling height is: H = m / (BD × π × R) 2 ) m - Mass of the dried soil after drying, in grams; BD - Soil bulk density (dry bulk density), g / cm³; R - Inner radius of soil simulation culture unit 201, cm; π - Pi (the mathematical constant in Chinese).

[0022] Traditional closed culture devices (such as wide-mouth plastic boxes, wide-mouth glass bottles) Figure 8 The existing closed cultivation device (as shown) suffers from two major technical bottlenecks: ① Physical structural instability: It cannot simulate and control the bulk density of natural soil, and the closed bottom structure easily leads to the formation of an anaerobic environment during long-term cultivation; ② Imbalanced water and air management: Long-term reliance on frequent artificial watering causes significant disturbance to the soil microenvironment and makes it difficult to maintain water stability, completely failing to simulate the continuous and stable capillary watering and free gas exchange process in nature. Therefore, to solve the problems of traditional closed cultivation devices, this invention makes the following improvements: 1. Core principle verification and modular design To verify the feasibility of the solution, prototype design and testing were conducted: "Soil Simulation Culture Unit 201" Principle Verification: By comparing direct soil filling with filling using a PVC ring cutter according to bulk density, it was confirmed that the latter can perfectly maintain the original structure and porosity of the soil. Figure 9 and Figure 10 (As shown).

[0023] Water vapor balance nested base 202 functional verification: Initially, the design was verified by comparing the bottom-sealed design with the design using nylon cloth sealing. Figure 9As shown in the figure, it is confirmed that nylon fabric can simultaneously achieve three major functions: water permeability, air permeability, and capillary water replenishment, effectively preventing anaerobic environments and realizing automatic and stable water supply.

[0024] 2. System Integration and Function Implementation After successfully verifying the above principles, the modules were integrated: the "soil simulation culture unit 201" was nested within the "water-air balance nested base 202," and the entire unit was placed in a glass bottle containing a small amount of water. The top was connected to the air path via a "multi-channel air path control valve 102." This system successfully constructed a standardized culture platform that is controllable, repeatable, and monitorable.

[0025] 3. Comprehensive performance verification and data support To quantitatively evaluate the technical effects of this invention, a one-month cultivation experiment was conducted. Figure 11 As shown in the figure, gas samples were collected at 0, 1, 3, 5, and 31 days, and measured using gas chromatography (6890A, GC, Agilent Technologies, USA) and isotope ratio mass spectrometry (Delta 12 Plus, Thermo Fisher Scientific, Germany) at different time points. 13 CO2 release was measured, and soil samples were destructively collected after 31 days. Fluorescence was measured using a multi-functional microplate reader (Scientific Fluoroskan Ascent FL, Thermo, USA), with excitation and emission wavelengths at 365 nm and 460 nm, respectively, to calculate soil enzyme activity. Microbial biomass carbon was determined using a total organic carbon analyzer (Vario TOC, Elementar, Germany). Soil pore structure characteristics after the incubation experiment were analyzed using X-ray micro-CT (Phoenix Nanotom X-ray μ-CT). Additionally, soil bulk density and soil microbial carbon use efficiency were measured and calculated. The technical advantages of this invention have been fully verified. Compared with traditional incubation devices (… Figure 8 Compared to traditional culture devices that place soil directly at the bottom (using a wide-mouth bottle), this invention demonstrates significant advantages in maintaining the stability of the soil ecosystem. Figure 2-7 ).

[0026] In terms of physical structure, the soil cultivation system of this invention effectively maintains the original state of the soil. After cultivation, the soil bulk density and porosity in the system of this invention are not significantly different from the initial state; conversely, traditional devices show a significant decrease in bulk density and an abnormal increase in porosity. Figure 2-3 This indicates that conventional devices may lead to the destruction of soil aggregate structure due to imbalanced water management, thereby compromising its physical stability, while the present invention successfully avoids this structural degradation.

[0027] In terms of bioactivity, the system of this invention provides a more suitable habitat for microbial communities. Soil cultured using conventional devices showed a significant decrease in both microbial biomass carbon and β-glucosidase activity, revealing the strong inhibition of anaerobic environments on total microbial populations and their key ecological functions. Figure 4-5 In contrast, the system of this invention successfully maintained MBC and BG enzyme activities similar to the initial state, demonstrating that its internal environment is conducive to the survival of the microbial community and the conduct of normal physiological activities.

[0028] The technical effects of this invention are particularly outstanding in terms of core metabolic functions and process monitoring. Microbial carbon utilization efficiency (CUE) is a key indicator for measuring microbial metabolic strategies. The sharp decrease in CUE in conventional devices indicates that their anaerobic environment forces microorganisms to consume more carbon substrates through respiration rather than for growth. Figure 6 This metabolic pathway is also directly reflected in the release dynamics of ¹³CO2: the cumulative release of traditional devices is not only lower, but also shows an abnormal trend of fluctuating decrease, reflecting the disorder of its metabolic activity; while the present invention shows a classic kinetic curve of steady increase, confirming its healthy internal aerobic metabolic process. Figure 7 ).

[0029] In summary, the above results constitute a complete chain of evidence, profoundly revealing the systemic defects of traditional closed-system culture devices. Their inability to maintain a stable water-air balance leads to the destruction of soil physical structure and triggers a series of biological chain reactions—from reduced microbial numbers and impaired functional enzyme activity to low core metabolic efficiency, ultimately resulting in inaccurate isotope tracing experimental data (…). 13 The CO2 release kinetics were severely distorted. In contrast, this invention, through biomimetic design, successfully simulated the natural soil-water-air exchange interface, maintaining the stability of the soil ecological microenvironment in all aspects during the one-month cultivation period. This provides a reliable technical platform for the precise quantification of key ecological processes such as soil carbon turnover. This not only demonstrates the inventiveness of the invention but also highlights its significant application value in improving the fidelity of soil culture experiments.

[0030] The soil culture system of this invention has also been used in other project experiments, but due to space limitations, it will not be described in detail here.

[0031] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A soil cultivation system simulating natural ecological conditions, characterized in that, The soil cultivation system includes an undisturbed soil structure maintenance structure (1), and a soil-water-air interface regulation structure (2) is provided inside the undisturbed soil structure maintenance structure (1). The original soil structure maintenance structure (1) includes a culture bottle (101), the upper end of the culture bottle (101) is provided with a bottle cap, a gas passage pipe is provided through the center of the bottle cap, and a multi-channel gas passage control valve (102) is provided on the gas passage pipe. The soil-water-air interface regulation structure (2) includes a soil simulation culture unit (201), and a water-air balance nested base (202) is provided at the lower end of the soil simulation culture unit (201). The water-air balance nested base (202) has multiple openings, and multiple rows of openings are provided on the side wall of the water-air balance nested base (202). The lower end of the water-air balance nested base (202) is wrapped with a water-permeable and air-permeable support membrane (203).

2. The soil cultivation system simulating natural ecological conditions according to claim 1, characterized in that, The soil simulation culture unit (201) is a PVC ring cutter with an inner diameter of 46 mm, a height of 60 mm, and a volume of 100 cm³. 3 .

3. The soil cultivation system simulating natural ecological conditions according to claim 1, characterized in that, The permeable and breathable support membrane (203) is made of nylon fabric with a pore size of 400 mesh; the water-air balance nested base (202) is made of PVC.

4. The soil cultivation system simulating natural ecological conditions according to claim 1, characterized in that, The water-air balance nested base (202) has 69 openings, and the side wall of the water-air balance nested base (202) has 4 rows of openings, with 3 openings in each row; the openings are circular, and the radius of the circle is 1 mm.

5. A soil cultivation method simulating natural ecological conditions, characterized in that, The soil cultivation method includes the following steps: The soil was filled to a height calculated according to the natural soil bulk density. Based on the filling height, the soil was divided into four layers and filled into the soil simulation culture unit (201). Each layer of soil was compacted and cleaned before filling the next layer. Then, a stable isotope marker was added to the surface of the soil simulation culture unit (201). The water-air balance nested base (202) was installed at the lower end of the soil simulation culture unit (201) filled with soil. Then, it was placed in the culture bottle (101). Ultrapure water was added to the bottom of the culture bottle (101). Ultrapure water was added along the inner wall of the culture bottle (101) every 20 days.

6. The soil cultivation method for simulating natural ecological conditions according to claim 5, characterized in that, The amount of soil used is 100 g; the compaction is done using a soil compactor; the cleaning is done using a PVC brush; and the cleaning time is 30 s.

7. The soil cultivation method for simulating natural ecological conditions according to claim 5, characterized in that, Add 2 mg of the stable isotope marker per gram of soil; add 5 mL of ultrapure water to the bottom of the culture flask (101), and add 5 mL of ultrapure water along the inner wall of the culture flask (101) every 20 days.

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