Method for improving soil carbon sink based on coupling of carbon dioxide nanobubble / chemical energy autotrophic microbial agent / low-release charcoal

Abstract

The invention belongs to the technical field of soil improvement, and particularly relates to a method for improving soil carbon sink based on carbon dioxide nanobubble / chemical energy autotrophic microbial agent / low-release charcoal coupling. The method comprises the following steps: mixing and stirring a chemoautotrophic microbial agent, low-release charcoal obtained by saccharifying and pyrolyzing biomass straws, and carbon dioxide nano bubble water in soil. Compared with the prior art, the method has the advantages that the organic carbon content of the soil can be greatly increased, and meanwhile, the soil pH is not reduced, the salt concentration is not increased, and the soil respiration is not greatly increased, that is, the soil acidification, the soil salinization and the increase of CO2 emission caused by the soil respiration are avoided. The method is a green, efficient and pollution-free soil improvement technology.

Description

Technical Field

[0001] This invention belongs to the field of soil improvement technology, specifically relating to a method for enhancing soil carbon sequestration based on the coupling of carbon dioxide nanobubbles / chemoautotrophic microbial agents / low-release biochar. Background Technology

[0002] With the increasing prominence of global climate change, enhancing soil carbon sequestration potential and increasing soil organic carbon storage have become important strategic directions for addressing climate change and improving soil quality. As the largest carbon pool in terrestrial ecosystems, soil's organic carbon content not only directly affects soil fertility, water and fertilizer retention capacity, and microbial activity, but also plays a crucial regulatory role in the global carbon cycle balance. Therefore, developing efficient and green soil improvement technologies to enhance soil carbon sequestration has become a research hotspot and urgent need in the field of agricultural ecology and environment.

[0003] Currently, there are many types of soil improvement technologies, mainly including physical, chemical, and biological methods. Physical improvement methods, such as deep plowing and straw return to the field, can improve soil structure to some extent, but their effect on increasing soil organic carbon is limited, and they suffer from problems such as being time-consuming, labor-intensive, and lacking sustainability. Chemical improvement methods mostly improve soil fertility by applying chemical fertilizers and humic acid substances, but long-term use can easily lead to soil salinization and acidification, damage the soil microbial community structure, and cause secondary environmental problems. At the same time, because many soil conditioners can increase soil respiration (i.e., increase CO2 release), their long-term promoting effect on soil carbon sequestration is not significant.

[0004] Biological soil improvement methods have gradually become a research focus in the field of soil improvement due to their advantages such as environmental friendliness and strong sustainability. Chemoautotrophic microorganisms can synthesize organic carbon by utilizing inorganic carbon sources, which theoretically can directly increase the organic carbon content of soil and enhance soil carbon sequestration. However, in practical applications, it has been found that most microbial agents, when directly added to the soil, are easily deactivated due to the complex soil environment (such as nutrient deficiency and competition from native microorganisms). Therefore, it is difficult to achieve a sustained increase in organic carbon by directly applying chemoautotrophic microbial agents to the soil.

[0005] To address the survival and efficacy issues of microbial inoculants in soil, researchers have attempted to couple their application with biochar. However, existing technologies primarily utilize traditional biochar, which readily releases soluble substances (such as small-molecule organic acids and ash) into the environment. These releases can interfere with the metabolic activities of chemoautotrophic bacteria and promote soil respiration, potentially inhibiting their carbon sequestration function and failing to meet the need for a significant increase in soil carbon sequestration.

[0006] Besides the need for optimization of microbial-biochar coupling technology, other existing novel single technologies also have significant shortcomings. For example, some technical solutions attempt to introduce nanobubble water technology to increase soil organic carbon content. For instance, a Chinese patent (application number: 202411031125.0) uses a mixed carbon dioxide and oxygen gas source to prepare nanobubbles, achieving a 30%-34% increase in soil organic carbon after 14 days of soil improvement. However, it did not assess the long-term effects of soil improvement, nor did it evaluate whether the nanobubble technology significantly affected soil respiration, microbial activity, pH, and dissolved oxygen. Furthermore, the application of combined technologies is nowhere to be seen.

[0007] In summary, current soil improvement technologies all have significant shortcomings in increasing soil organic carbon storage: single technologies have limited effectiveness in increasing soil organic carbon and lack sustainability; existing improvement technologies also have negative effects such as soil salinization, acidification, decreased dissolved oxygen, and abnormally increased soil respiration. Therefore, developing a soil improvement technology that can effectively enhance soil carbon sequestration, is environmentally friendly, and has no negative effects has become an urgent technical challenge to be solved in this field. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a method for enhancing soil carbon sequestration based on the coupling of carbon dioxide nanobubbles, chemoautotrophic microbial agents, and low-release biochar.

[0009] The objective of this invention can be achieved through the following technical solutions: A method for enhancing soil carbon sequestration based on the coupling of carbon dioxide nanobubbles / chemoautotrophic microbial agents / low-release biochar includes the following steps: S1. Mix the soil with low-release biochar evenly to obtain a biochar-soil mixture; S2. After activating and culturing the chemoautotrophic microbial agent, centrifuge the mixture and collect the bacterial precipitate. S3. Add the bacterial precipitate obtained in step S2 to diluted carbon dioxide nanobubble water and mix to prepare a microbial suspension. S4. Pour the microbial suspension obtained in step S3 into the carbon-soil mixture obtained in step S1.

[0010] Furthermore, the low-release biochar is produced by burning the solid residue from biomass straw after saccharification at a high temperature of 300-700°C under anaerobic conditions. Compared with ordinary biochar, this saccharified biochar achieves a significant reduction (more than 85%) in the amount of soluble released substances.

[0011] Furthermore, the saccharification process is as follows: placing biomass straw in an acidic aqueous solution, adding cellulase to carry out enzymatic hydrolysis, and after enzymatic hydrolysis, centrifuging and drying to obtain the solid residue; The acidic aqueous solution is either citric acid solution or sulfuric acid solution; the pH value of the acidic aqueous solution is 4.8~5.2. The solid-liquid ratio of the biomass straw to the acidic aqueous solution is 1g:(10~20)mL; preferably, the solid-liquid ratio of the biomass straw to the acidic aqueous solution is 1g:10mL. The ratio of cellulase to biomass straw is 2~300 FPU / g; The enzymatic hydrolysis temperature is 40~45℃, and the enzymatic hydrolysis time is 40~55 h.

[0012] Preferably, the enzymatic hydrolysis temperature is 45°C and the enzymatic hydrolysis time is 48 h.

[0013] Furthermore, the chemoautotrophic microbial agent is composed of... Ochrobactrum sp. WH–2 (CICC 23802), Stenotrophomonas sp. (CICC 23803), Ochrobactrum sp. WH–13 (CICC 23804), Castellaniella sp. WH–14 (CICC 23805) or Sinomicrobium oceani WH–15 (CICC23806) was prepared by mixing at least three strains in equal mass ratios; all strains are preserved at the China Industrial Microbial Culture Collection Center (CICC).

[0014] Further, in step S2, the activation and cultivation process of the chemoautotrophic microbial agent is as follows: the chemoautotrophic microbial agent is first cultured in LB culture medium for 20-30 h at 30-35℃, then transferred to LB culture medium for 10-15 h for activation and recovery, and finally transferred to inorganic culture medium for acclimatization and cultivation for 45-50 h; after the cultivation is completed, the supernatant is discarded by centrifugation and the bacterial precipitate is collected.

[0015] Furthermore, the LB culture medium is prepared by adding water to LB broth medium, and its final concentration is 25 g / L; The inorganic culture medium comprises: (NH4)2SO4 5.0 g / L, KH2PO4 1.0 g / L, K2HPO4 2.0 g / L, MgSO4·7H2O 0.2 g / L, NaCl 20 g / L, CaCl2 0.01 g / L and FeSO4·7H2O 0.01 g / L, with 2 mL of trace element solution added per liter of the inorganic culture medium. The trace element solution comprises the following components: Na₂MoO₄·2H₂O 1.68 mg / L, H₃BO₃ 0.4 mg / L, ZnSO₄·7H₂O 1.0 mg / L, MnSO₄·5H₂O 1.0 mg / L, CuSO₄·5H₂O 7.0 mg / L, CoCl₂·6H₂O 1.0 mg / L, and NiSO₄·7H₂O 1.0 mg / L.

[0016] Furthermore, the inorganic culture medium is filtered through a 220 nm filter membrane before use to remove microorganisms; the acclimatization culture is carried out in a serum bottle, and the serum bottle is filled with carbon dioxide gas, such that the volume of carbon dioxide occupies 10-30% of the volume of the serum bottle, preferably 20%.

[0017] Furthermore, the carbon dioxide nanobubble water is prepared by a nanobubble generator, and the volume percentage of carbon dioxide in the gas source introduced during the preparation process is not less than 10%, with the remainder being any inert gas or air; the pH value of the carbon dioxide nanobubble water is less than 6.

[0018] The diluted carbon dioxide nanobubble water mentioned in step S3 is prepared by mixing and diluting carbon dioxide nanobubble water with water, wherein the volume percentage of carbon dioxide nanobubble water in the diluted system is 8% to 20%.

[0019] Further, the mixing mass ratio of the soil and low-release biochar in step S1 is 100:(0.5~2). The microbial suspension OD in step S3 600 The optical density value at a wavelength of 600 nm is controlled between 0.3 and 0.6. In step S4, the solid-liquid ratio of the charcoal mixture to water after irrigation is 1 g: (0.25~0.30) mL (based on the dry weight of the charcoal mixture).

[0020] Furthermore, it also includes the following steps: After completing step S4, once the soil enters the slow dehydration stage, add an equal amount of the diluted carbon dioxide nanobubble water according to the water loss reduction of the initial carbon soil mixture sample weight.

[0021] Preferably, the diluted carbon dioxide nanobubble water is watered once every 7 days. After 7 days of cultivation, the soil goes through a rapid dehydration stage and then enters a slow dehydration stage. At this time, the soil moisture content is low and the proportion of free water decreases significantly. By regularly replenishing the carbon dioxide nanobubble water, the stable operation of the coupling system can be maintained.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention constructs a novel soil carbon sequestration enhancement technology system by coupling the application of chemoautotrophic microbial agents and carbon dioxide nanobubble water, combined with the enhancement and protection of microbial function by low-release biochar. Through the coupled effect of chemoautotrophic microbial agents, carbon dioxide nanobubble water, and low-release biochar, a technical effect of "1+1+1>3" is achieved. After several months of application to soil, this technology can significantly increase soil organic carbon content without causing problems such as decreased soil pH, increased salt concentration, or a dramatic increase in soil respiration rate. It fundamentally avoids the risks of soil acidification, salinization, and additional carbon dioxide emissions. It is a green, efficient, and pollution-free soil improvement technology, providing a new technical solution and theoretical basis for solving global soil ecological environment problems and climate change issues. The synergistic mechanism and core advantages of the three-part coupling are specifically reflected in the following aspects: The chemoautotrophic microbial agent used in this invention is a compound of various autotrophic microorganisms with efficient carbon dioxide fixation function. It can synthesize organic carbon from gaseous carbon dioxide through biotransformation without generating any additional pollutants, thus possessing both environmental friendliness and sustainability. The diluted carbon dioxide nanobubble water not only provides a sufficient and stable inorganic carbon source for carbon-fixing microorganisms in the soil, but also maintains the continuity of carbon source supply through the slow-release characteristics of nanobubbles. This effectively stimulates and enhances the carbon dioxide fixation activity of microorganisms, significantly improving the efficiency of soil organic carbon generation and solving the problems of insufficient carbon source supply and limited carbon fixation efficiency of single agents.

[0023] The diluted carbon dioxide nanobubble water can restore the pH and dissolved oxygen content to the level of conventional water bodies, effectively regulating the local dissolved oxygen state of the soil, significantly reducing the probability of soil acidification and oxygen stress, and creating a suitable survival environment for microorganisms. The low-release biochar used in conjunction with it has the characteristics of low release content and stable performance. It can adsorb and fix some nanobubbles with its porous structure, prolonging the contact time between the carbon source and microorganisms, and avoiding secondary pollution such as soil salinization caused by traditional biochar releases from the source. At the same time, it provides a colonization carrier for microorganisms and improves the survival stability of the inoculant in the soil. The two work together to solve the shortcomings of single technologies that are prone to causing soil microenvironment imbalance and microbial activity decline.

[0024] Low-release biochar, combined with chemoautotrophic microbial agents and carbon dioxide nanobubble water, forms a functionally enhanced cycle, amplifying the overall carbon sequestration and carbon sink effects. Low-release biochar not only protects microorganisms from the complex soil environment, but its stable physicochemical properties also promote the efficient conversion of carbon sources provided by nanobubble water by microorganisms, reducing the decomposition and loss of organic carbon. Simultaneously, the organic carbon generated by microbial conversion can be further adsorbed and fixed by biochar, improving the soil's organic carbon sequestration efficiency and forming a complete closed loop of "carbon source supply - bioconversion - carbon sink sequestration." Compared to single technologies or simple combination of two technologies, this significantly improves the stability and longevity of soil carbon sequestration. Attached Figure Description

[0025] Figure 1 The soil organic carbon content and soil respiration value after 3 months of cultivation in Example 1 and Comparative Examples 1-5 of this invention; Figure 2 The soil electrical conductivity and pH value after 3 months of cultivation in Example 1 and Comparative Example 5 of this invention; Figure 3 This refers to the sustained-release effect of carbon dioxide nanobubble water used in Example 1 of the present invention; Figure 4 The graph shows the changes in pH and dissolved oxygen under different dilution ratios of sparkling water. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0027] Unless otherwise specified, the raw materials and equipment used in this invention are all commercially available products and conventional equipment.

[0028] Example 1 A method for enhancing soil carbon sequestration based on the coupling of carbon dioxide nanobubbles, chemoautotrophic microbial agents, and low-release biochar, comprising the following specific steps: S1. Place crop straw powder in a citric acid aqueous solution with pH=5, add cellulase and mix evenly. Perform enzymatic hydrolysis at 45℃ for 48 h. After enzymatic hydrolysis, centrifuge and dry to obtain solid residue. Then, burn the solid residue at 500℃ in an oxygen-free environment to obtain low-release biochar. Mix the soil with the above low-release biochar at a mass ratio of 100:1 to obtain a biochar-soil mixture. The solid-liquid ratio of the crop straw powder to the acidic aqueous solution is 1g:10mL; the material ratio of the cellulase to the crop straw powder is 200 FPU / g.

[0029] S2. The chemoautotrophic microbial agent was first cultured in LB medium at 30℃ for 24 h, then transferred to fresh LB medium for 12 h for activation and recovery. Finally, the recovered bacterial solution was transferred to a serum bottle containing inorganic medium and acclimatized for 48 h. During the acclimatization period, carbon dioxide gas was injected into the serum bottle to fill 20% of the serum bottle volume. The bacterial precipitate was collected. After the culture was completed, the system was centrifuged at 8000 rpm for 6 min, the supernatant was discarded, and the bacterial precipitate was collected.

[0030] The chemoautotrophic microbial agent is prepared by mixing five strains in equal mass ratio from the China Industrial Microbial Culture Collection Center (CICC). The names and preservation numbers of the five strains are as follows: Ochrobactrum sp. WH–2 (CICC 23802), Stenotrophomonas sp. (CICC 23803), Ochrobactrum sp. WH–13 (CICC 23804), Castellaniella sp. WH–14 (CICC 23805) or Sinomicrobium oceani WH–15 (CICC 23806); The LB culture medium was prepared by adding water to LB broth medium, and its final concentration was 25 g / L. The inorganic culture medium consists of the following components: (NH4)2SO4 5.0 g / L, KH2PO4 1.0 g / L, K2HPO4 2.0 g / L, MgSO4·7H2O 0.2 g / L, NaCl 20 g / L, CaCl2 0.01 g / L and FeSO4·7H2O 0.01 g / L, with 2 mL of trace element solution added per liter of inorganic culture medium. The trace element solution comprises: Na₂MoO₄·2H₂O 1.68 mg / L, H₃BO₃ 0.4 mg / L, ZnSO₄·7H₂O 1.0 mg / L, MnSO₄·5H₂O 1.0 mg / L, CuSO₄·5H₂O 7.0 mg / L, CoCl₂·6H₂O 1.0 mg / L, and NiSO₄·7H₂O 1.0 mg / L. The inorganic culture medium is filtered through a 220 nm filter membrane to remove microorganisms before use.

[0031] S3. Start the shear-type nanobubble generator (Southern Pump Industry Co., Ltd., model 20QY-1), and introduce 100% high-purity carbon dioxide gas as the gas source into the device. Under the conditions of water pressure 0.1 MPa and gas flow rate 0.1 L / min, continue to prepare for 10 min to obtain carbon dioxide nanobubble water. Then, mix and dilute the obtained carbon dioxide nanobubble water with water at a volume ratio to obtain diluted carbon dioxide nanobubble water (wherein the volume percentage of carbon dioxide nanobubble water in the diluted system is 10%). Take the bacterial precipitate collected in step S2, add it to the above diluted carbon dioxide nanobubble water, mix thoroughly to obtain a microbial suspension, and control the OD of the microbial suspension. 600 It is 0.4.

[0032] S4. The microbial suspension obtained in step S3 is poured into the carbon-soil mixture obtained in step S1. After pouring, the solid-liquid ratio of the carbon-soil mixture to water is 1 g: 0.30 mL (based on the dry weight of the carbon-soil mixture).

[0033] S5. After completing step S4, every 7 days, add an equal amount of diluted carbon dioxide nanobubble water prepared in step S3, based on the amount of water loss from the initial dry weight of the carbon-soil mixture sample.

[0034] After 3 months of cultivation, the organic carbon content, soil respiration, pH value and electrical conductivity data in the soil were measured.

[0035] Comparative Example 1 A method for enhancing soil carbon sequestration based on chemoautotrophic microbial inoculants / low-release biochar, the specific steps of which are as follows: S1. Place crop straw powder in a citric acid aqueous solution with pH=5, add cellulase and mix evenly. Perform enzymatic hydrolysis at 45℃ for 48 h. After enzymatic hydrolysis, centrifuge and dry to obtain solid residue. Then, burn the solid residue at 500℃ in an oxygen-free environment to obtain low-release biochar. Mix the soil with the above low-release biochar at a mass ratio of 100:1 to obtain a biochar-soil mixture. The solid-liquid ratio of the crop straw powder to the acidic aqueous solution is 1g:10mL; the material ratio of the cellulase to the crop straw powder is 200 FPU / g.

[0036] S2. The chemoautotrophic microbial agent was first cultured in LB medium at 30℃ for 24 h, then transferred to fresh LB medium for 12 h for activation and recovery. Finally, the recovered bacterial solution was transferred to a serum bottle containing inorganic medium and acclimatized for 48 h. During the acclimatization period, carbon dioxide gas was injected into the serum bottle to fill 20% of the serum bottle volume. The bacterial precipitate was collected. After the culture was completed, the system was centrifuged at 8000 rpm for 6 min, the supernatant was discarded, and the bacterial precipitate was collected.

[0037] The chemoautotrophic microbial agent is prepared by mixing five strains in equal mass ratio from the China Industrial Microbial Culture Collection Center (CICC). The names and preservation numbers of the five strains are as follows: Ochrobactrum sp. WH–2 (CICC 23802), Stenotrophomonas sp. (CICC 23803), Ochrobactrum sp. WH–13 (CICC 23804), Castellaniella sp. WH–14 (CICC 23805) or Sinomicrobium oceani WH–15 (CICC 23806); The LB culture medium was prepared by adding water to LB broth medium, and its final concentration was 25 g / L. The inorganic culture medium consists of the following components: (NH4)2SO4 5.0 g / L, KH2PO4 1.0 g / L, K2HPO4 2.0 g / L, MgSO4·7H2O 0.2 g / L, NaCl 20 g / L, CaCl2 0.01 g / L and FeSO4·7H2O 0.01 g / L, with 2 mL of trace element solution added per liter of inorganic culture medium. The trace element solution comprises: Na₂MoO₄·2H₂O 1.68 mg / L, H₃BO₃ 0.4 mg / L, ZnSO₄·7H₂O 1.0 mg / L, MnSO₄·5H₂O 1.0 mg / L, CuSO₄·5H₂O 7.0 mg / L, CoCl₂·6H₂O 1.0 mg / L, and NiSO₄·7H₂O 1.0 mg / L. The inorganic culture medium is filtered through a 220 nm filter membrane to remove microorganisms before use.

[0038] S3. Take the bacterial precipitate collected in step S2, add it to water, mix thoroughly to obtain a microbial suspension, and control the OD of the microbial suspension. 600 It is 0.4.

[0039] S4. The microbial suspension obtained in step S3 is poured into the carbon-soil mixture obtained in step S1. After pouring, the solid-liquid ratio of the carbon-soil mixture to water is 1 g: 0.30 mL (based on the dry weight of the carbon-soil mixture).

[0040] S5. After completing step S4, every 7 days, add an equal amount of water according to the amount of water loss from the initial dry weight of the carbon-soil mixture sample.

[0041] After 3 months of cultivation, the organic carbon content in the soil and changes in soil respiration were measured.

[0042] Comparative Example 2 A method for enhancing soil carbon sequestration based on carbon dioxide nanobubbles / low-release biochar, the specific steps of which are as follows: S1. Place crop straw powder in a citric acid aqueous solution with pH=5, add cellulase and mix evenly. Perform enzymatic hydrolysis at 45℃ for 48 h. After enzymatic hydrolysis, centrifuge and dry to obtain solid residue. Then, burn the solid residue at 500℃ in an oxygen-free environment to obtain low-release biochar. Mix the soil with the above low-release biochar at a mass ratio of 100:1 to obtain a biochar-soil mixture. The solid-liquid ratio of the crop straw powder to the acidic aqueous solution is 1g:10mL; the material ratio of the cellulase to the crop straw powder is 200 FPU / g.

[0043] S2. Start the shear-type nanobubble generator (Southern Pump Industry Co., Ltd., model 20QY-1), and introduce 100% high-purity carbon dioxide gas into the device as the gas source. Under the conditions of water pressure 0.1 MPa and gas flow rate 0.1 L / min, continue to prepare for 10 min to obtain carbon dioxide nanobubble water. Then, mix and dilute the obtained carbon dioxide nanobubble water with water according to the volume ratio to obtain diluted carbon dioxide nanobubble water (wherein the volume ratio of carbon dioxide nanobubble water in the diluted system is 10%).

[0044] S3. Pour the diluted carbon dioxide nanobubble water obtained in step S2 into the carbon-soil mixture obtained in step S1. After pouring, the solid-liquid ratio of the carbon-soil mixture to water is 1 g: 0.30 mL (based on the dry weight of the carbon-soil mixture).

[0045] S4. After completing step S3, every 7 days, add an equal amount of diluted carbon dioxide nanobubble water prepared in step S2, based on the amount of water loss from the initial dry weight of the carbon-soil mixture sample.

[0046] After 3 months of cultivation, the organic carbon content in the soil and changes in soil respiration were measured.

[0047] Comparative Example 3 A method for enhancing soil carbon sequestration based on carbon dioxide nanobubbles / chemoautotrophic microbial agents is presented. The specific steps are basically the same as in Example 1, except that the low-release biochar preparation and biochar-soil mixture preparation process in step S1 are removed; the original soil is directly used to replace the biochar-soil mixture, and subsequent steps are performed as in Example 1. The specific steps are as follows: S1. The chemoautotrophic microbial agent was first cultured in LB medium at 30℃ for 24 h, then transferred to fresh LB medium and cultured for 12 h for activation and recovery. Finally, the recovered bacterial solution was transferred to a serum bottle containing inorganic culture medium and acclimatized for 48 h. During the acclimatization period, carbon dioxide gas was injected into the serum bottle to fill 20% of the serum bottle volume. The bacterial precipitate was collected. After the culture was completed, the system was centrifuged at 8000 rpm for 6 min, the supernatant was discarded, and the bacterial precipitate was collected.

[0048] The chemoautotrophic microbial agent is prepared by mixing five strains in equal mass ratio from the China Industrial Microbial Culture Collection Center (CICC). The names and preservation numbers of the five strains are as follows: Ochrobactrum sp. WH–2 (CICC 23802), Stenotrophomonas sp. (CICC 23803), Ochrobactrum sp. WH–13 (CICC 23804), Castellaniella sp. WH–14 (CICC 23805) or Sinomicrobium oceani WH–15 (CICC 23806); The LB culture medium was prepared by adding water to LB broth medium, and its final concentration was 25 g / L. The inorganic culture medium consists of the following components: (NH4)2SO4 5.0 g / L, KH2PO4 1.0 g / L, K2HPO4 2.0 g / L, MgSO4·7H2O 0.2 g / L, NaCl 20 g / L, CaCl2 0.01 g / L and FeSO4·7H2O 0.01 g / L, with 2 mL of trace element solution added per liter of inorganic culture medium. The trace element solution comprises: Na₂MoO₄·2H₂O 1.68 mg / L, H₃BO₃ 0.4 mg / L, ZnSO₄·7H₂O 1.0 mg / L, MnSO₄·5H₂O 1.0 mg / L, CuSO₄·5H₂O 7.0 mg / L, CoCl₂·6H₂O 1.0 mg / L, and NiSO₄·7H₂O 1.0 mg / L. The inorganic culture medium is filtered through a 220 nm filter membrane to remove microorganisms before use.

[0049] S2. Start the shear-type nanobubble generator (Southern Pump Industry Co., Ltd., model 20QY-1), and introduce 100% high-purity carbon dioxide gas as the gas source into the device. Under the conditions of water pressure 0.1 MPa and gas flow rate 0.1 L / min, continue to prepare for 10 min to obtain carbon dioxide nanobubble water. Then, mix and dilute the obtained carbon dioxide nanobubble water with water at a volume ratio to obtain diluted carbon dioxide nanobubble water (wherein the volume percentage of carbon dioxide nanobubble water in the diluted system is 10%). Take the bacterial precipitate collected in step S1, add it to the above diluted carbon dioxide nanobubble water, mix thoroughly to obtain a microbial suspension, and control the OD of the microbial suspension. 600 It is 0.4.

[0050] S3. The microbial suspension obtained in step S2 is poured into the soil sample. After pouring, the solid-liquid ratio of soil to water is 1g: 0.30mL (based on the dry weight of soil).

[0051] S4. After completing step S3, every 7 days, add an equal amount of diluted carbon dioxide nanobubble water prepared in step S3, based on the amount of water loss from the initial soil sample's dry weight.

[0052] After 3 months of cultivation, the organic carbon content in the soil and changes in soil respiration were measured.

[0053] Comparative Example 4 A method for enhancing soil carbon sequestration based on carbon dioxide nanobubbles, the specific steps of which are as follows: S1. Start the shear-type nanobubble generator (Southern Pump Industry Co., Ltd., model 20QY-1), and introduce 100% high-purity carbon dioxide gas as the gas source into the device. Under the conditions of water pressure 0.1 MPa and gas flow rate 0.1 L / min, continue to prepare for 10 min to obtain carbon dioxide nanobubble water. Then, mix and dilute the obtained carbon dioxide nanobubble water with water according to the volume ratio to obtain diluted carbon dioxide nanobubble water (wherein the volume ratio of carbon dioxide nanobubble water in the diluted system is 10%).

[0054] S2. The diluted carbon dioxide nanobubble water obtained in step S1 is poured into the soil sample. After pouring, the solid-liquid ratio of soil to water is 1 g: 0.30 mL (based on the dry weight of soil).

[0055] S3. After completing step S2, every 7 days, add an equal amount of diluted carbon dioxide nanobubble water according to the amount of water loss from the initial soil dry weight.

[0056] After 3 months of cultivation, the organic carbon content in the soil and changes in soil respiration were measured.

[0057] Comparative Example 5 The blank control method for irrigating soil with pure water is as follows: Irrigate the soil with pure water and stir evenly, and control the solid-liquid ratio of soil to water after irrigation to be 1 g: 0.30 mL (based on soil dry weight); subsequently, every 7 days, add an equal amount of water according to the water loss from the initial soil dry weight.

[0058] After 3 months of cultivation, the organic carbon content, soil respiration, pH value and electrical conductivity data in the soil were measured.

[0059] The organic carbon content in the soils of the above embodiments and comparative examples was determined using a total organic carbon analyzer (model TOC-VCPH, Shimadzu, Japan); soil respiration changes were measured using a soil respiration meter (model LI-8100A, LI-COR, USA); soil electrical conductivity was determined using a conductivity meter (model FE38, Mettler Toledo Instruments Ltd., Shanghai, China); and soil pH was determined using a portable multi-parameter three-channel analyzer (model HQ4300, Hach, USA).

[0060] Figure 1 The soil organic carbon content and soil respiration value after 3 months of cultivation in Examples 1 and Comparative Examples 1-5 are shown. The results show that the soil organic carbon content of the three-technology coupling treatment (Example 1) reached 35.3 mg / g, which is 17.3 mg / g higher than that of the low-release biochar and inoculant coupling treatment (Comparative Example 1), an increase of 96.4%; 12.8 mg / g higher than that of the carbon dioxide nanobubble and low-release biochar coupling treatment (Comparative Example 2), an increase of 56.9%; 24.4 mg / g higher than that of the carbon dioxide nanobubble and chemoautotrophic microbial inoculant coupling treatment (Comparative Example 3), an increase of 224.3%; 24.6 mg / g higher than that of carbon dioxide nanobubble water alone (Comparative Example 4), an increase of 228.1%; and 25.9 mg / g higher than that of the original soil (Comparative Example 5, blank control), an increase of 273.2%. Soil organic carbon levels three months after soil improvement showed that the carbon sequestration effect of the three-technology coupling treatment was significantly better than all comparative examples, and its soil organic carbon content was higher than the sum of the contents of comparative examples 1 and 4, directly demonstrating the synergistic carbon sequestration effect of this coupling technology. Soil respiration rate is a commonly used indicator to characterize the intensity of carbon dioxide release by microbial respiration, reflecting soil microbial activity and soil carbon release characteristics. Compared with comparative examples 1-5, the soil respiration rate of Example 1 did not show an increasing trend, but rather showed a certain decreasing characteristic. This indicates that the three-technology coupling of the present invention is a green carbon sequestration technology, which achieves efficient accumulation of soil organic carbon by promoting carbon dioxide fixation by soil autotrophic microorganisms, rather than increasing the soil carbon pool by enhancing microbial heterotrophic metabolism. In summary, Figure 1Data shows that the carbon dioxide nanobubbles / chemoautotrophic microbial agents / low-release biochar coupling system can significantly increase the soil organic carbon content by 273.2% compared with the original blank soil, while also effectively inhibiting soil respiration and reducing the release of soil carbon dioxide.

[0061] Soil electrical conductivity is a commonly used indicator to measure the degree of soil salinization. This invention measured the electrical conductivity data of Example 1 and a blank soil sample (Comparative Example 5). Figure 2 As shown, the electrical conductivity of the blank soil (Comparative Example 5) was 209.6 μS / cm, while the electrical conductivity of the soil in Example 1 was 178.0 μS / cm. Compared with the blank soil, the electrical conductivity of the soil in Example 1 decreased by 15.1%. This indicates that the three-technology coupling treatment of the present invention did not cause or aggravate soil salinization after 3 months of soil improvement, and may even show a decreasing trend. In addition, the soil pH of Example 1 and Comparative Example 5 were 8.3 and 8.4, respectively, which are basically close, indicating that Example 1 did not have a significant impact on soil pH. This verifies that the application of the three-technology coupling to soil can not only continuously improve carbon sequestration but also avoid causing secondary environmental pollution.

[0062] Figure 3 The slow-release effect of the carbon dioxide nanobubble water used in Example 1 is illustrated. The contents of inorganic carbon (IC) and organic carbon were measured using a total organic carbon analyzer (TOC-VCPH, Shimadzu, Japan); the number of nanobubbles was detected using a nanoparticle tracking analyzer (ZetaView PMX 120 BASIC, Particle Metrix, Germany). Over a 14-day observation period, the inorganic carbon content in the water gradually decreased, while the number of nanobubbles continuously decreased. This indicates that the carbon dioxide nanobubble water can slowly release carbon dioxide into the soil environment, thereby continuously stimulating and promoting the carbon fixation function of autotrophic microorganisms in the soil. This phenomenon fully demonstrates the unique advantages of nanobubbles: long retention time; in contrast, ordinary bubbles (with a diameter in the millimeter range) in existing technologies only have a retention time of a few hours in water.

[0063] Figure 4The graph shows the pH and dissolved oxygen changes at different dilution ratios of the carbon dioxide nanobubble water. Both indicators were measured using a portable multi-parameter three-channel analyzer (model HQ4300, Hach, USA). This graph illustrates the design basis for diluting the carbon dioxide nanobubble water to 10% in Example 1 of this invention. As can be seen from the graph, 100% concentration carbon dioxide nanobubble water is acidic (pH < 6). The acidity of the solution gradually decreases with increasing dilution. When diluted to 10%, its pH value is basically consistent with that of conventional water. This indicates that using 10% concentration carbon dioxide nanobubble water, compared to pure carbon dioxide nanobubble water (100% concentration), can significantly reduce the likelihood of soil acidification. Furthermore, the dissolved oxygen content of 10% concentration carbon dioxide nanobubble water is close to that of conventional water, while high concentrations of carbon dioxide can reduce dissolved oxygen content to some extent. Therefore, diluting the carbon dioxide nanobubble water to 10% can effectively avoid soil acidification and oxygen stress problems.

[0064] In summary, this invention, through the coupling of three technologies—carbon dioxide nanobubbles, chemoautotrophic microbial agents, and low-release biochar—can significantly increase soil organic carbon content without exacerbating soil respiration. Furthermore, addressing potential environmental risks such as salinization, acidification, and oxygen stress arising from the application of these technologies, this invention has undergone targeted optimization at both the initial design and application levels, substantially reducing the probability of such problems occurring. Whether considering soil improvement effectiveness or environmental safety, this invention has a positive promoting effect on soil improvement.

[0065] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for improving soil carbon sink based on carbon dioxide nanobubble / chemoautotrophic microbial inoculant / low-release biochar coupling, characterized in that, The method comprises the following steps: S1, mixing the soil with low-release biochar uniformly to obtain a carbon-soil mixture; S2, after the activation culture of the chemotrophic autotrophic microbial agent, centrifugal treatment is performed to collect the bacterial precipitate; S3, the bacterial precipitate obtained in step S2 is added into diluted carbon dioxide nano-bubble water to mix and prepare a microbial suspension; S4, the microbial suspension obtained in step S3 is irrigated into the carbon-soil mixture obtained in step S1.

2. The method for improving soil carbon sink based on carbon dioxide nanobubble / chemoautotrophic microbial inoculant / low-release biochar coupling according to claim 1, characterized in that, The low-release biochar is prepared by high-temperature calcination of solid residues generated by saccharification treatment of biomass straw under anaerobic conditions at 300-700 DEG C.

3. The method for improving soil carbon sink based on carbon dioxide nanobubble / chemoautotrophic microbial inoculant / low-release biochar coupling according to claim 2, characterized in that, The saccharification treatment process is that the biomass straw is placed in an acidic aqueous solution, cellulase is added for enzymatic hydrolysis reaction, and after the enzymatic hydrolysis is completed, the solid residues are obtained by centrifugation and drying; The acidic aqueous solution is a citric acid aqueous solution or a sulfuric acid solution; the pH value of the acidic aqueous solution is 4.8-5.2; The solid-liquid ratio of the biomass straw to the acidic aqueous solution is 1g:(10-20)mL; The material ratio of the cellulase to the biomass straw is 2-300 FPU / g; The enzymatic hydrolysis temperature is 40-45 DEG C, and the enzymatic hydrolysis time is 40-55 h.

4. The method for enhancing soil carbon sink based on carbon dioxide nanobubble / chemoautotrophic microbial inoculant / low-release biochar coupling according to claim 1, characterized in that, The chemolithotrophic microbial agent is prepared by mixing at least three strains of bacteria in equal mass ratio, wherein the bacteria are selected from the group consisting of Ochrobactrum Pseudomonas sp. WH-2 (CICC 23802), Stenotrophomonas Pseudomonas sp. (CICC 23803), Ochrobactrum Pseudomonas sp. WH-13 (CICC 23804), Castellaniella Pseudomonas sp. WH-14 (CICC 23805), or Sinomicrobium oceani Pseudomonas sp. WH-15 (CICC 23806).

5. The method for enhancing soil carbon sink based on carbon dioxide nanobubble / chemoautotrophic microbial inoculant / low-release biochar coupling according to claim 1, characterized in that, In step S2, the activation culture process of the chemotrophic autotrophic microbial agent is that the chemotrophic autotrophic microbial agent is first cultured in LB culture solution for 20-30 h at 30-35 DEG C, then transferred into LB culture solution for 10-15 h for activation and recovery, and finally transferred into inorganic culture solution for domestication culture for 45-50 h; after the culture is completed, the supernatant is discarded by centrifugation, and the bacterial precipitate is collected.

6. The method for enhancing soil carbon sink based on carbon dioxide nanobubble / chemoautotrophic microbial inoculum / low-release biochar coupling according to claim 5, characterized in that, The LB culture solution is prepared by adding water to LB bouillon culture medium, and the final concentration is 25 g / L; The components of the inorganic culture solution include (NH4)2SO4 5.0 g / L, KH2PO4 1.0 g / L, K2HPO4 2.0 g / L, MgSO4·7H2O 0.2 g / L, NaCl 20 g / L, CaCl2 0.01 g / L, and FeSO4·7H2O 0.01 g / L, and 2 mL of trace element solution is added per liter of the inorganic culture solution; The components of the trace element solution include Na2MoO4·2H2O 1.68 mg / L, H3BO3 0.4 mg / L, ZnSO4·7H2O 1.0 mg / L, MnSO4·5H2O 1.0 mg / L, CuSO4·5H2O 7.0 mg / L, CoCl2·6H2O 1.0 mg / L, and NiSO4·7H2O 1.0 mg / L.

7. The method for enhancing soil carbon sink based on carbon dioxide nanobubble / chemolithoautotrophic microbial inoculant / low-release biochar coupling according to claim 5, characterized in that, The inorganic culture solution is filtered through a 220 nm filter membrane to remove microorganisms before use; the acclimation culture is carried out in a serum bottle, and the serum bottle is filled with carbon dioxide gas, so that the volume of carbon dioxide accounts for 10-30% of the volume of the serum bottle.

8. The method for enhancing soil carbon sink based on carbon dioxide nanobubble / chemolithoautotrophic microbial inoculant / low-release biochar coupling according to claim 1, characterized in that, The carbon dioxide nanobubble water is prepared by a nanobubble generating device, and the volume percentage of carbon dioxide in the gas source introduced during preparation is not less than 10%; The diluted carbon dioxide nanobubble water in step S3 is obtained by mixing and diluting carbon dioxide nanobubble water with water, and the volume percentage of carbon dioxide nanobubble water in the system after dilution is 8-20%.

9. The method for enhancing soil carbon sink based on carbon dioxide nanobubble / chemolithoautotrophic microbial inoculant / low-release biochar coupling according to claim 1, characterized in that, The mixing mass ratio of the soil to the low-release biochar in step S1 is 100:(0.5-2); The OD of the microbial bacteria suspension in step S3 600 Control at 0.3-0.6; In step S4, the solid-liquid ratio of the carbon-soil mixture to water after irrigation is 1 g:(0.25-0.30) mL.

10. The method for enhancing soil carbon sink based on carbon dioxide nanobubble / chemolithoautotrophic microbial inoculant / low-release biochar coupling according to claim 1, characterized in that, Further comprising the step: after completing step S4, when the soil enters the slow dehydration stage, according to the water loss reduction amount of the initial carbon-soil mixture sample weight, an equal amount of the diluted carbon dioxide nanobubble water is added.

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