Soil carbon sequestration-pollution reduction-yield increase multi-effect synergistic microbial agent and application thereof in improvement of ploughing ability

Abstract

The invention relates to the technical field of soil conditioners and applications thereof, and discloses a soil carbon sequestration-pollution reduction-yield increase multi-effect synergistic microbial inoculum and an application thereof in ploughing ability improvement, the soil carbon sequestration-pollution reduction-yield increase multi-effect synergistic microbial inoculum comprises an organic pollutant degradation functional flora, a soil carbon sequestration functional flora and a small molecular organic matter production functional flora; the organic pollutant degrading functional flora comprises efficient pollutant degrading bacteria; the soil carbon sequestration functional flora comprises ammonia oxidizing bacteria, ammonia oxidizing archaea, complete ammonia oxidizing bacteria, sulfate reducing bacteria, iron reducing bacteria, rhizobium and photosynthetic flora; the micromolecular organic matter production functional flora comprises heterotrophic nitrification-aerobic denitrification bacteria, zoogloea, phosphorus-accumulating bacteria and photosynthetic bacteria. According to the scheme, multiple florae are specifically arranged in the synergistic agent, and through the synergistic interaction effect of the florae, the effects of the synergistic agent in multiple fields of soil carbon sequestration, pollution reduction and yield increase are effectively improved.

Description

Technical Field

[0001] This invention relates to the field of soil conditioners and their application technology, specifically to a multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase, and its application in improving arable land capacity. Background Technology

[0002] Currently, existing technologies for soil pollution remediation mostly employ physical and chemical remediation methods. Physical remediation methods, such as soil replacement and deep tillage, are costly and can easily damage soil structure. While chemical remediation methods are faster-acting, they may introduce new chemicals, causing secondary pollution, and have limited effect on improving soil fertility. Regarding soil carbon sequestration, measures such as increasing the application of organic fertilizers and returning straw to the field are mainly used, but these methods have relatively slow carbon sequestration effects and are difficult to coordinate with soil pollution remediation and crop yield increase. In terms of promoting crop yield, the large-scale application of chemical fertilizers is mainly relied upon, which not only exacerbates soil pollution and carbon pool instability but also leads to a decline in the quality of agricultural products. Therefore, existing technologies cannot achieve a multi-effect synergy of soil carbon sequestration, pollution reduction, and crop yield increase, and cannot fundamentally solve the comprehensive problems currently facing farmland soils. How to effectively coordinate the production functions, pollution control, and carbon sequestration capacity of the arable land system to achieve a multi-effect synergy of increased yield, pollution reduction, and carbon sequestration is crucial for ensuring food security and promoting the transformation and innovation of farmland protection. Summary of the Invention

[0003] The present invention aims to provide a multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction and yield increase and its application in improving arable land capacity, so as to solve the technical problem that existing microbial agents are unable to achieve multi-effect synergy of soil carbon sequestration, pollution reduction and crop yield increase.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: a multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase, comprising a functional microbial community for degrading organic pollutants, a functional microbial community for soil carbon sequestration, and a functional microbial community for producing small molecule organic matter; the functional microbial community for degrading organic pollutants includes highly efficient pollutant-degrading bacteria; the functional microbial community for soil carbon sequestration includes ammonia-oxidizing bacteria, ammonia-oxidizing archaea, complete ammonia-oxidizing bacteria, sulfate-reducing bacteria, iron-reducing bacteria, rhizobia, and photosynthetic bacteria; the functional microbial community for producing small molecule organic matter includes heterotrophic nitrifying-aerobic denitrifying bacteria, bacterial flocs, polyphosphate-accumulating bacteria, and photosynthetic bacteria.

[0005] The principle and advantages of this scheme are: This solution enhances the effectiveness of synergistic microbial agents in multiple areas of soil carbon sequestration, pollution reduction, and yield increase through their synergistic effects. Specifically, addressing the high concentrations of organic pollutants such as phthalates (PAEs) and polycyclic aromatic hydrocarbons (PAHs) in soil, this solution incorporates organic pollutant-degrading microbial communities into the synergistic agent, effectively degrading these pollutants and reducing their levels. Addressing the issue of low soil carbon sequestration capacity, this solution includes soil carbon-sequestration microbial communities in the synergistic agent. These communities convert atmospheric CO2 or organic carbon into stable soil carbon pools through metabolic activities, effectively improving soil carbon sequestration capacity and increasing soil carbon sequestration volume. Addressing the severe loss of soil organic matter, this solution includes small-molecule organic matter-producing microbial communities in the synergistic agent. The polysaccharides, amino acids, humic substances, and polypeptides produced by these microbial communities significantly enhance soil nutrients.

[0006] 1. Multifunctional Synergy to Enhance Soil Capacity: This solution combines the addition of pollutant-degrading bacteria to rapidly mineralize organic toxins, carbon-fixing bacteria (ammonia-oxidizing bacteria / iron-reducing bacteria / photosynthetic bacteria) to simultaneously fix CO2 and passivate heavy metals, and heterotrophic nitrification-aerobic denitrification bacteria and polyphosphate-accumulating bacteria to efficiently convert nitrogen and phosphorus nutrients, achieving an integrated approach of "pollution removal-carbon sequestration-fertility enhancement" and significantly improving soil health.

[0007] 2. Long-lasting effect, assisting in optimizing soil self-recovery capacity: This solution, in conjunction with the addition of photosynthetic bacteria, continuously provides energy to drive microbial metabolism, and the bacterial flocs enhance the biofilm's stress resistance. Combined with the nutrient cycle of polyphosphate-accumulating bacteria + heterotrophic nitrification-aerobic denitrification bacteria, a self-sustaining microecology is formed, reducing the need for repeated application and extending the remediation effect to more than 3 times that of conventional microbial agents.

[0008] 3. Improve fertilizer utilization and effectively increase yield: This solution, in conjunction with microbial agents, shortens the nitrogen removal pathway by adding heterotrophic nitrifying-aerobic denitrifying bacteria, thereby significantly improving nitrogen utilization. Polyphosphate-accumulating bacteria activate unavailable phosphorus in the soil, and combined with nitrogen fixation by rhizobia and root promotion by photosynthetic bacteria, it increases yield while degrading pollution, and ensures that grain heavy metals meet standards.

[0009] Preferably, as an improvement, the raw materials include the following parts by weight: 5-8 parts of ammonia-oxidizing bacteria, 5-8 parts of ammonia-oxidizing archaea, 5-8 parts of complete ammonia-oxidizing bacteria, 2-5 parts of sulfate-reducing bacteria, 2-6 parts of iron-reducing bacteria, 5-11 parts of rhizobia, 22-30 parts of highly efficient pollutant-degrading bacteria, 20-25 parts of heterotrophic nitrification-aerobic denitrification bacteria, 8-14 parts of bacterial flocs, 3-6 parts of polyphosphate-accumulating bacteria, and 6-12 parts of photosynthetic bacteria.

[0010] Technical Effects: This solution, employing the above-mentioned setup, avoids the ineffectiveness of functional microbial communities due to insufficient dosage (e.g., traditional microbial agents with carbon-fixing bacteria <10 parts) and prevents resource waste (e.g., polyphosphate-accumulating bacteria >6 parts would compete for carbon sources with photosynthetic bacteria). This design enables the microbial agent to achieve triple benefits of carbon fixation, increased production, and pollution reduction while reducing the cost of contaminated soil remediation by 40%. Specifically, firstly, high dosage of core functional microbial communities ensures the main efficacy: highly efficient pollutant-degrading bacteria (22-30 parts) and heterotrophic nitrification-aerobic denitrification bacteria (20-25 parts) account for over 50%, ensuring rapid mineralization and simultaneous denitrification of organic pollutants; photosynthetic bacteria (6-12 parts) provide sufficient energy to drive the carbon fixation chain, supporting the long-term operation of the microbial community. Secondly, the gradient compatibility and synergistic effect of carbon-fixing and passivation microbial communities: ammonia-oxidizing bacteria (AOB / AOA / Comammox, 5-8 parts each) account for 15-24 parts, covering the entire pH / oxygen environment to achieve NH4+. + Zero accumulation; sulfate-reducing bacteria (2-5 parts) + iron-reducing bacteria (2-6 parts) form a "sulfur-iron dual pathway": low doses effectively precipitate heavy metals (sulfides) and adsorb oxidized toxins (iron oxides), avoiding excessive reduction leading to soil acidification. Secondly, the precise ratio of auxiliary microbial communities enhances ecological resilience: bacterial flocs (8-14 parts) account for about 10%, secreting sufficient EPS to encapsulate functional bacteria, effectively improving stress resistance; while rhizobia (5-11 parts) and polyphosphate-accumulating bacteria (3-6 parts) form a "nitrogen and phosphorus supply unit", with the nitrogen fixation capacity of rhizobia (maximum of 11 parts) matching the phosphorus solubilization capacity of polyphosphate-accumulating bacteria (maximum of 6 parts), meeting 80% of the crop's nitrogen and phosphorus needs. Finally, photosynthetic bacteria are used across species: they are both carbon-fixing bacteria (6-12 parts) and small-molecule production bacteria, playing a dual role to reduce the total bacterial load.

[0011] Preferably, as an improvement, the following raw materials are preferred in parts by weight: 6 parts ammonia-oxidizing bacteria, 6 parts ammonia-oxidizing archaea, 6 parts complete ammonia-oxidizing bacteria, 4 parts sulfate-reducing bacteria, 3 parts iron-reducing bacteria, 6 parts rhizobia, 25 parts highly efficient pollutant-degrading bacteria, 22 parts heterotrophic nitrifying-aerobic denitrifying bacteria, 10 parts bacterial flocs, 4 parts polyphosphate-accumulating bacteria, and 8 parts photosynthetic bacteria.

[0012] Technical Effects: This solution employs the above-mentioned setup, facilitating the full utilization of each component and achieving synergistic effects. Specifically, a high dose of pollution-reducing bacteria (25 parts highly efficient degrading bacteria + 22 parts HN-AD bacteria, totaling 47%) rapidly removes organic pollutants, while simultaneously, a precisely formulated carbon-fixing bacterial community (18 parts ammonia-oxidizing bacteria + 8 parts photosynthetic bacteria + 3 parts iron-reducing bacteria) effectively eliminates NH4 derived from pollutants. + Zero accumulation and CO2 mineral sequestration; supplemented by stress resistance-fertilizer supply module (10 parts of bacterial flocs to enhance survival rate, 6 parts of rhizobium + 4 parts of polyphosphate-accumulating bacteria to optimize nitrogen and phosphorus supply), forming a self-circulating system of "degradation-carbon sequestration-yield increase", ultimately achieving a synergistic effect of PAHs and PAEs in pesticides reaching a degradation rate of over 92% within 3 days, increasing soil carbon sequestration, ensuring crop heavy metal compliance and increasing yield.

[0013] Preferably, as an improvement, the ammonia-oxidizing bacteria include Nitrosospira_multiformis , Nitrosococcus_oceani , Nitrosomonas_europaea One or more combinations of the following; ammonia-oxidizing archaea include Nitrosopumilus_maritimus and / or Nitrososphaera_gargensis Complete ammonia-oxidizing bacteria include Candidatus_Nitrospiranitrosa and / or Candidatus_Nitrospiranitrificans Sulfate-reducing bacteria include Pseudomonas_sp. HLS-6 and / or Bacillus cereus Iron-reducing bacteria include Shewanella_ one-eyed , Geobacter_sp. , Acidimicrobium One or more combinations of the following; rhizobia include Agrobacterium , Azorhizobium , Bradyrhizobium , Devosia_geojensis One or more combinations thereof.

[0014] Technical Effects: The ammonia-oxidizing bacteria in this scheme function to: autotrophically utilize ammonia and assimilate inorganic substances such as carbon dioxide. The ammonia-oxidizing archaea function to: autotrophically utilize ammonia and assimilate inorganic substances such as carbon dioxide. The fully ammonia-oxidizing bacteria function to: directly oxidize ammonia nitrogen to nitrate and degrade organic matter. The sulfate-reducing bacteria function to: assimilate carbon dioxide, reduce sulfate, and remove organic pollutants and heavy metals. The iron-reducing bacteria function to: assimilate carbon dioxide, degrade macromolecular organic matter, and reduce heavy metal ions to a low-toxicity, low-valence state. The rhizobia function to: fix nitrogen symbiotically with leguminous plants and transport nitrogen for plant utilization.

[0015] Preferably, as an improvement, the photosynthetic bacteria include Rhodopseudomonas_palustris and / or Rhodopseudomonas_faecalis .

[0016] Technical effect: The role of the above-mentioned photosynthetic bacteria in this solution is to use carbon dioxide to perform photosynthesis to synthesize their own substances, form small molecule organic matter, and produce hydrogen, fix nitrogen, and decompose organic matter.

[0017] Preferably, as an improvement, the highly efficient pollutant-degrading bacteria include Enterobacter_cloacae , Saccharomonospora_azurea , Nocardiopsis_alba , Sphingomomonas_yanoikuyae One or more combinations thereof.

[0018] Technical effect: The role of the above-mentioned highly efficient pollutant degrading bacteria in this solution is to degrade phthalates (PAEs) and polycyclic aromatic hydrocarbons (naphthalene, fluorene, phenanthrene and pyrene).

[0019] Preferably, as an improvement, the heterotrophic nitrifying-aerobic denitrifying bacteria include Acinetobacter_ Baumann's , Faecal_alkaligenes , Paracoccus_denitrificans , Microbacterium_ shell , Thiobacillus_denitrificans One or more combinations of the following; fungal micelles include Zoogloea_ rice and / or Mitsuaria_chitosanitabida Polyphosphate-accumulating bacteria include Tetrasphaera_elongata and / or Paenibacillus_ lautus CL-5 .

[0020] Technical Effects: The heterotrophic nitrifying-aerobic denitrifying bacteria used in this solution degrade organic pollutants and produce small-molecule nutrients that are easily absorbed by plants. The bacterial flocs provide a living environment for other microorganisms, promoting their functions; adsorb and oxidize organic matter; remove heavy metal pollution; and bind the organic matter produced by the microbial community to the soil. The polyphosphate-accumulating bacteria enhance the efficiency of phosphate fertilizers, converting solid phosphorus in the soil into a form available to plants, reducing the amount of phosphate fertilizer required; and remove excess phosphorus introduced by pesticides.

[0021] Preferably, as an improvement, this solution also provides a method for preparing a multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase, comprising the following steps: Step 1: Based on the organic pollutant degradation functional bacteria, add heterotrophic nitrifying-aerobic denitrifying bacteria, bacterial flocs, and rhizobia in sequence according to the proportion, mix well and record it as mixture number one; Step 2: Mix sulfate-reducing bacteria and iron-reducing bacteria in a certain proportion, then add ammonia-oxidizing bacteria, ammonia-oxidizing archaea, and complete ammonia-oxidizing bacteria in sequence. Mix well and let stand for 30-60 minutes. This mixture is called Mixture No. 2. Step 3: Mix Mixture 1 and Mixture 2 1-2 hours before use, add photosynthetic bacteria, place under natural light, and let stand still to obtain synergistic bacterial agent.

[0022] Technical effects: This solution adopts the above settings and uses a step-by-step mixing strategy to precisely match functional microbial communities, avoiding mutual inhibition among microbial communities; it utilizes photosynthetic bacteria as an energy engine to drive carbon fixation, while coupling microbial micelles to enhance stress resistance, achieving synergistic effects of efficient degradation of organic pollutants, in-situ passivation of heavy metals, nitrogen emission reduction and soil carbon fixation, ultimately increasing crop yield while forming a self-sustaining soil micro-ecological cycle, significantly extending the effective period and reducing application costs.

[0023] Preferably, as an improvement, this solution also provides an application of a multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase in improving arable land capacity, including the following application method: before application, the soil is loosened to control the total porosity of the soil to 45~65%, the aeration porosity to >10%, and the soil moisture content to >15%; then the synergistic microbial agent prepared within 5 hours is applied to the soil surface and then tilled to the soil layer within 20cm.

[0024] Technical Effects: This solution, employing the aforementioned setup, controls the total soil porosity to 45-65%, aeration porosity >10%, and moisture content >15% by loosening the soil before application. This creates a suitable microenvironment for the functional microorganisms in the microbial agent, ensuring the activity of aerobic microorganisms for efficient carbon sequestration and pollutant degradation. Simultaneously, the "fresh application" method, allowing application within 5 hours, reduces the loss of microbial agent activity. Combined with tilling to the inner 20cm soil layer, the microbial agent directly acts on the core area with dense crop roots, achieving a multi-effect synergistic effect of carbon sequestration (converting active carbon into stable organic carbon), pollution reduction (accelerating the degradation of surface pollutants), and yield increase (improving rhizosphere nutrient utilization). Furthermore, it is easy to operate and adaptable to various types of arable land, providing both short-term benefits and long-term improvement in soil structure and fertility, thus enhancing the overall capacity of arable land. Attached Figure Description

[0025] Figure 1 This is a diagram illustrating the process of PAHs degradation by the multi-effect synergistic microbial agent CFPRM-1 in Example 1 of the present invention.

[0026] Figure 2 This is a process diagram of the degradation of PAEs by the multi-effect synergistic microbial agent CFPRM-1 in Example 1 of the present invention, which is used for soil carbon sequestration, pollution reduction, and yield increase.

[0027] Figure 3 The degradation rate of PAHs and PAEs by the multi-effect synergistic microbial agent CFPRM-1 for soil carbon sequestration, pollution reduction, and yield increase in Example 1 of this invention within 72 h. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0029] Overview of the Plan This solution provides a multi-functional synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield enhancement, comprising microbial communities for degrading organic pollutants, microbial communities for soil carbon sequestration, and microbial communities for producing small molecule organic matter. The microbial communities for degrading organic pollutants include highly efficient pollutant-degrading bacteria. The microbial communities for soil carbon sequestration include ammonia-oxidizing bacteria, ammonia-oxidizing archaea, fully ammonia-oxidizing bacteria, sulfate-reducing bacteria, iron-reducing bacteria, rhizobia, and photosynthetic bacteria. The microbial communities for producing small molecule organic matter include heterotrophic nitrifying-aerobic denitrifying bacteria, bacterial flocs, polyphosphate-accumulating bacteria, and photosynthetic bacteria.

[0030] The raw materials include the following parts by weight: 5-8 parts ammonia-oxidizing bacteria, 5-8 parts ammonia-oxidizing archaea, 5-8 parts complete ammonia-oxidizing bacteria, 2-5 parts sulfate-reducing bacteria, 2-6 parts iron-reducing bacteria, 5-11 parts rhizobia, 22-30 parts highly efficient pollutant-degrading bacteria, 20-25 parts heterotrophic nitrification-aerobic denitrification bacteria, 8-14 parts bacterial flocs, 3-6 parts polyphosphate-accumulating bacteria, and 6-12 parts photosynthetic bacteria.

[0031] The following raw materials are preferred in parts by weight: 6 parts ammonia-oxidizing bacteria, 6 parts ammonia-oxidizing archaea, 6 parts complete ammonia-oxidizing bacteria, 4 parts sulfate-reducing bacteria, 3 parts iron-reducing bacteria, 6 parts rhizobia, 25 parts highly efficient pollutant-degrading bacteria, 22 parts heterotrophic nitrifying-aerobic denitrifying bacteria, 10 parts bacterial flocs, 4 parts polyphosphate-accumulating bacteria, and 8 parts photosynthetic bacteria. The classification, names, and preservation information of the bacterial species in each functional microbial community used in this scheme are shown in Table 1, and their culture characteristics are shown in Table 2.

[0032] Table 1. Strain Information

[0033] Table 2. Culture characteristics of each bacterial group

[0034] Specifically, the composition of various functional microbial communities and the culture methods of each strain are as follows: (1) Soil carbon sequestration functional microbial community 1-1. Ammonia-oxidizing bacteria include Nitrosospira_multiformis , Nitrosococcus_oceani , Nitrosomonas_europaea One or more combinations of the following; ammonia-oxidizing archaea include Nitrosopumilus_maritimus and / or Nitrososphaera_gargensis Complete ammonia-oxidizing bacteria include Candidatus_Nitrospiranitrosa and / or Candidatus_Nitrospiranitrificans .

[0035] Furthermore, ammonia-oxidizing bacteria, ammonia-oxidizing archaea, and complete ammonia-oxidizing bacteria are cultured using the following steps: After the bacterial strains were activated, ammonia-oxidizing bacteria and ammonia-oxidizing archaea were directly cultured in pure form. Complete ammonia-oxidizing bacteria were cultured in domestic sewage diluted 5 times, with ammonia-oxidizing inorganic salt culture medium and trace element solution added. The culture was then allowed to stand or stirred at low speed under the conditions of temperature 28~30℃, pH 7.5~8.0, and DO 1.5~3.0mg / L.

[0036] The composition of the ammonia-oxidized inorganic salt culture medium is as follows: per 1 L of culture medium, it includes (NH4)2SO4: 0.5~1.0 g, NaHCO3: 1.0~1.5 g, KH2PO4: 0.1 g, MgSO4·7H2O: 0.05 g, CaCl2·2H2O: 0.01 g, pH: 7.5~8.2.

[0037] The composition of the trace element solution is as follows: based on 1L of culture medium, it includes C 10 H 14 N2Na2O8: 5.0g, FeSO4·7H2O: 2.0g, ZnSO4·7H2O: 0.1g, MnCl2·4H2O: 0.03g, CoCl2·6H2O: 0.2g, NiCl2·6H2O: 0.02g, Na2MoO4·2H2O: 0.03g.

[0038] 1-2. Sulfate-reducing bacteria include Pseudomonas_sp. HLS-6 and / or Bacillus cereus The following steps are used for cultivation: Sulfate-reducing bacteria culture medium was used, and the bacteria were cultured in an anaerobic workstation (O2 < 0.1%) at a temperature of 25-32℃ and a pH of 7.5-8.0.

[0039] The composition of the sulfate-reducing bacteria culture medium is as follows: per 1L of medium, it includes Na2SO4: 4.5g (electron acceptor), C3H5NaO3: 3.5g (electron donor), NH4Cl: 1.0g, KH2PO4: 0.5g, MgSO4·7H2O: 2.0g, CaCl2·2H2O: 0.06g, FeSO4·7H2O: 0.004g, yeast extract: 1.0g, and ascorbic acid: 0.1g.

[0040] 1-3. Iron-reducing bacteria include Shewanella_oneidensis , Geobacter_sp. , Acidimicrobium One or more combinations thereof; cultured using the following steps: Use iron-reducing bacteria culture medium with 1 mL / L trace element solution, and use cysteine-HCl (0.5~1 mM) or Na2S (0.1~0.2 mM) as reducing agents. Culture in an anaerobic workstation at a temperature of 25~32℃ and a pH of 7.5~8.0.

[0041] The composition of the iron-reducing bacteria culture medium is as follows: per 1L of medium, it includes sodium acetate (NaC2H3O2): 10~20mM, sodium lactate (C3H5NaO3): 10~20mM, amorphous Fe(III) oxide: 10~50mM, ferric citrate (FeC6H5O7): 5~10mM, NaHCO3: 2~5g, HEPES or PIPES buffer: 10~20mM, NH4Cl: 0.5~1.0g, KH2PO4: 0.1~0.2g, and KCl: 0.1g.

[0042] 1-4. Rhizobia include Agrobacterium , Azorhizobium , Bradyrhizobium , Devotions_ george's One or more combinations thereof; cultured using the following steps: Yeast mannitol medium was used, air was introduced, and the culture was carried out at a speed of 120-150 rpm, a temperature of 25-32℃, and a pH of 7.5-8.0.

[0043] The yeast mannitol culture medium consists of the following components per 1 L of medium: mannitol: 10.0 g, yeast extract: 0.4 g, K2HPO4: 0.5 g, MgSO4·7H2O: 0.2 g, NaCl: 0.1 g, and CaCl2·2H2O: 0.1 g.

[0044] 1-5. Photosynthetic bacteria include Rhodopseudomonas_palustris and / or Rhodopseudomonas fecal The following steps are used for cultivation: Use purple non-sulfur bacterial culture medium, add 1 mL / L trace element solution (composition as described above), control the light intensity to 1000~3000 lux, micro-aerobic (DO<0.5mg / L), and culture at a temperature of 25~35℃, a shaking speed of 150-170 rpm, and a pH of 6.5~7.5.

[0045] The composition of the purple non-sulfur bacteria culture medium is as follows: per 1L of culture medium, it includes C2H3NaO2: 1.0~3.0g or C4H6O5: 2.0~5.0g, (NH4)2SO4: 1.0~2.0g or yeast extract: 0.5~1.0g, KH2PO4: 0.5g, MgSO4·7H2O: 0.2g, and NaCl: 0.5~2.0g.

[0046] (2) Microbial community for degrading organic pollutants: 2-1. Highly efficient pollutant-degrading bacteria include Enterobacter_cloacae , Saccharomonospora azure , Nocardiopsis_alba , Sphingomomonas_yanoikuyae One or more combinations thereof; cultured using the following steps: Use phthalic acid ester (PAE) degrading bacteria culture medium, add 1 mL / L trace element solution, aerate, and culture at 120-150 rpm, 25-32℃, and pH 7.5-8.0.

[0047] The composition of the phthalic acid ester (PAE) degrading bacteria culture medium is as follows: per 1L of culture medium, it includes Na2HPO4: 2.5g, KH2PO4: 1.0g, NH4Cl: 1.0g, MgSO4·7H2O: 0.2g, CaCl2: 0.01g, FeSO4·7H2O: 0.005g, and PAE contaminants: 10~100mg / L.

[0048] (3) Functional microbial communities for the production of small molecule organic matter: 3-1. Heterotrophic nitrifying-aerobic denitrifying bacteria include Acinetobacter_baumannii , Alkaligenes fecal , Paracoccus_denitrificans , Microbacterium_testaceum , Thiobacillus_ denitrifying One or more combinations thereof; cultured using the following steps: Use heterotrophic nitrifying-aerobic denitrifying bacteria culture medium, add 1 mL / L trace element solution, aerate, and culture at 150~200 rpm, 25~35℃, and pH 7.0~7.5.

[0049] The heterotrophic nitrifying-aerobic denitrifying bacteria culture medium consists of the following components per 1L of medium: sodium acetate (C2H3NaO2): 5-10g or sodium succinate (C4H4Na2O4): 5-10g, (NH4)2SO4: 0.5-1.0g or KNO3: 0.5-1.0g, KH2PO4: 1.0g, MgSO4·7H2O: 0.2g, NaCl: 0.5g, and CaCl2·2H2O: 0.01g.

[0050] 3-2. Flocs include Zoogloea_oryzae and / or Mitsuaria_chitosanitabida The following steps are used for cultivation: Use a bacterial floc culture medium, add 1 mL / L of trace element solution, aerate, and culture at a speed of 150-170 rpm, a temperature of 25-30℃, and a pH of 6.5-7.0.

[0051] The composition of the bacterial floc culture medium is as follows: per 1L of culture medium, it includes glucose or sucrose: 1.0–5.0g, (NH4)2SO4: 0.1~0.5g, KH2PO4: 0.5g, MgSO4·7H2O: 0.2g, CaCl2·2H2O: 0.05g, and NaCl: 0.1g.

[0052] 3-3. Polyphosphate-accumulating bacteria include Tetrasphaera_elongata and / or Paenibacillus_ lautus CL-5 The following steps are used for cultivation: Use polyphosphate-accumulating bacteria culture medium, add 1 mL / L trace element solution, control anaerobic-aerobic alternation (2-4 hours each), anaerobic DO <0.2 mg / L (nitrogen purging or sealing), aerobic DO 2-4 mg / L (aeration or shaking at 150 rpm), and culture at 20-30℃ and pH 7.0-7.5.

[0053] The composition of the polyphosphate-accumulating bacteria culture medium is as follows: per 1L of culture medium, it includes C2H3NaO2: 5~10mM, C3H5NaO2: 5~10mM, KH2PO4: 10~50mgP / L, NH4Cl: 20~50mgN / L, MgSO4·7H2O: 0.2g, CaCl2·2H2O: 0.05g, KCl: 0.1g, and NaHCO3: 0.5g.

[0054] After culturing using the methods described in this protocol, the viable count of each bacterial group was >1*10⁻⁶. 8 CFU / mL.

[0055] This solution also provides a method for preparing a multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase. The method involves preparing the raw materials for the synergistic agent in advance, including the following steps: Step 1: Based on the organic pollutant degradation functional bacteria, add heterotrophic nitrifying-aerobic denitrifying bacteria, bacterial flocs, and rhizobia in sequence according to the proportion, mix well and record it as mixture number one; Step 2: Mix sulfate-reducing bacteria and iron-reducing bacteria in a certain proportion, then add ammonia-oxidizing bacteria, ammonia-oxidizing archaea, and complete ammonia-oxidizing bacteria in sequence. Mix well and let stand for 30-60 minutes. This mixture is called Mixture No. 2. Step 3: Mix Mixture 1 and Mixture 2 1-2 hours before use, add photosynthetic bacteria, place under natural light, and let stand still to obtain synergistic bacterial agent.

[0056] This solution also provides an application of a multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase in improving arable land capacity. The following application methods are included: Before application, the soil is loosened to control the total porosity of the soil to 45-65%, the aeration porosity to >10%, and the soil moisture content to >15%; then the synergistic microbial agent prepared within 5 hours is applied to the soil surface at a rate of 1-3 L / acre, and then tilled to the soil layer within 20cm.

[0057] Example 1 In this embodiment, the multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase is CFPRM-1, which includes the following raw materials in parts by weight: 6 samples of ammonia-oxidizing bacteria ( Nitrosospira_multiformis ), 6 samples of ammonia-oxidizing archaea ( Nitrosopumilus_ maritime ), 6 complete ammonia oxidizing bacteria ( Candidatus_Nitrospiranitrosa ), 4 samples of sulfate-reducing bacteria ( Pseudomonas_sp. HLS-6 ), 3 iron-reducing bacteria ( Shewanella_oneidensis ), 6 samples of rhizobium ( Agrobacterium and Devosia _geojensis (Equal amounts mixed), 25 portions of highly efficient pollutant-degrading bacteria ( Enterobacter_cloacae and, Saccharomonospora_azurea (Equal amounts mixed), 22 portions of heterotrophic nitrifying-aerobic denitrifying bacteria ( Acinetobacter_baumannii and Faecal_alkaligenes Mix in equal amounts), 10 parts of mycelial flocs ( Zoogloea_oryzae ), 4 samples of polyphosphate () Tetrasphaera_elongata ), 8 samples of photosynthetic bacteria ( Rhodopseudomonas_palustris ).

[0058] Example 2 In this embodiment, the multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase is CFPRM-2, which includes the following raw materials in parts by weight: 5 samples of ammonia-oxidizing bacteria ( Nitrosococcus_oceani ), 7 samples of ammonia-oxidizing archaea ( Nitrososphere_ (gargensis) 7 samples of complete ammonia oxidizing bacteria ( Candidatus_Nitrospiranitrificans ), 3 samples of sulfate-reducing bacteria ( Bacillus ), 4 iron-reducing bacteria (A cicidimicrobium ), 7 samples of rhizobium ( Azorhizobium and Bradyrhizobium (Equal amounts mixed), 27 samples of highly efficient pollutant-degrading bacteria ( Nocardiopsis_alba and Sphingomomonas_yanoikuyae (Equal amounts mixed), 21 portions of heterotrophic nitrifying-aerobic denitrifying bacteria ( Microbacterium_testaceum and Thiobacillus_ denitrifying Mix in equal amounts), 12 portions of mycelial flocs ( Mitsuaria_chitosanitabida ), 3 samples of polyphosphate (POPs) Paenibacillus_lautus CL-5 ), 11 photosynthetic bacteria ( Rhodopseudomonas_faecalis ).

[0059] Example 3 In this embodiment, the multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase is CFPRM-3, which includes the following raw materials in parts by weight: 7 samples of ammonia-oxidizing bacteria ( Nitrosomonas_europaea ), 5 samples of ammonia-oxidizing archaea ( Nitrosopumilus_ maritime and Nitrososphaera_gargensis Mix equal amounts), 5 portions of complete ammonia oxidizing bacteria ( Candidate_ Nitrospiranitrosa and Nitrospiranitrifying candidate (Mix equal amounts) 5 parts sulfate-reducing bacteria Pseudomonas_sp. HLS-6 and Bacillus 6 iron-reducing bacteria ( Geobacter_sp.) 10 samples of rhizobium ( Agrobacterium and Azorhizobium (Equal amounts mixed), 23 samples of highly efficient pollutant-degrading bacteria ( Enterobacter_ sewers and Nocardiopsis_alba (Equal amounts mixed), 24 portions of heterotrophic nitrifying-aerobic denitrifying bacteria ( Alkaligenes_ fecal , Paracoccus_denitrificans and Microbacterium_testaceum Mix in equal amounts), 11 portions of mycelial flocs ( Zoogloea_oryzae and Mitsuaria_chitosanitabida Mix equal amounts), 6 portions of polyphosphate-accumulating bacteria ( Tetrasphaera_elongata and Paenibacillus_lautus CL-5 (Equal amounts mixed), 7 portions of photosynthetic bacteria ( Rhodopseudomonas_palustris and Rhodopseudomonas_faecalis Mix equal amounts.

[0060] The synergistic bacterial agent was obtained by referring to the preparation method described above.

[0061] Experimental Example 1: The effect of applying the synergistic microbial agent CFPRM-1 The application method described above was used in Example 1, which was applied to oil-contaminated rice soil at a rate of 1.7 L / mu.

[0062] First, for details on the degradation effect of the synergistic microbial agent CFPRM-1 on organic pollutants, please refer to [link to relevant documentation]. Figure 1~Figure 3 .

[0063] The results showed that the synergistic microbial agent CFPRM-1 achieved a degradation rate of over 95% for naphthalene (100 mg / L), fluorene (50 mg / L), phenanthrene (100 mg / L), and pyrene (50 mg / L) in PAHs within 72 h (e.g., Figure 1 As shown). The bacterial community CFPRM-1 achieved a degradation rate exceeding 92% for dibutyl phthalate (DBP), di(2-ethylhexyl) phthalate (DEHP), and dicyclohexyl phthalate (DCHP) in PAEs within 72 h (as shown). Figure 2~Figure 3 (As shown).

[0064] Secondly, the soil carbon sequestration potential before and after application of the synergistic microbial agent CFPRM-1 is shown in Table 3.

[0065] Table 3 Comparison of soil carbon sequestration capacity before and after application of synergistic microbial agent CFPRM-1

[0066] Experimental data show that the synergistic microbial agent CFPRM-1 significantly increased the content of soil SOM, TOC, DOC, and MBC. Especially 30 days after the application of the microbial agent, the strains cooperated with each other to effectively maintain the soil's carbon sequestration capacity.

[0067] Then, the changes in soil dissolved substance concentration (specifically, dissolved organic matter (DOM) concentration) after applying the synergistic microbial agent CFPRM-1 are shown in Table 4.

[0068] Table 4. Changes in dissolved organic matter in soil after application of microbial agent CFPRM-1

[0069] Experimental results show that the application of microbial agents greatly increased the concentration of DOM, and the microorganisms could maintain their activity and continuously metabolize to produce DOM and other substances for plant absorption and utilization.

[0070] The synergistic microbial agents in Examples 2-3 and Comparative Examples 1-4 are basically the same as those in Example 1, with the differences detailed in Table 5.

[0071] Table 5. Composition of synergistic microbial agents in Examples 2-3 and Comparative Examples 1-4

[0072] Experimental Example 2: Application Effects of Other Synergistic Microbial Agents Examples 2-3 and Comparative Examples 1-4 all obtained synergistic microbial agents according to the preparation methods described above, and applied them to petroleum-contaminated tomato soil according to the application methods described above. The application rate can be selected as 1-3 L / acre. The soil condition was tested after 30 days, and the results are detailed in Table 6.

[0073] Table 6. Effects of synergistic microbial agents on application in Examples 2-3 and Comparative Examples 1-4 (calculated 10 days after application).

[0074] Experimental data shows that CFPRM-1 bacterial agent needs to be fully mixed to exert its maximum effect; the absence of any part will greatly reduce the effectiveness of the bacterial agent.

[0075] The culture environments for the strains involved in this scheme include autotrophic and heterotrophic, aerobic, anaerobic, and facultative aerobic, and both light-required and light-independent environments. Autotrophic bacteria quickly lose their original functions under conditions rich in organic matter, and anaerobic bacteria also quickly lose their functions under high concentrations of dissolved oxygen (DO). Pollutant-degrading bacteria and bacterial flocs cannot function effectively at low organic matter concentrations. Furthermore, industry professionals generally believe that simply mixing strains can lead to antagonism and competition between bacterial communities, resulting in strain inactivation, reduced function, or even loss of function. This scheme, by optimizing the dosage of each strain, effectively reduces the antagonistic and competitive effects between multiple bacterial species and achieves synergistic effects among multiple bacterial communities. The absence of a certain functional bacterial community (e.g., comparative examples 1-3) or photosynthetic bacteria (e.g., comparative example 4) will significantly reduce the soil-improving effect of the synergistic agent.

[0076] In addition, this method mixes various bacterial groups in stages according to their respective characteristics, and then mixes them again 1-2 hours before use to add photosynthetic bacteria. This can effectively reduce the antagonism and competition between bacterial groups, and also allow various strains to fully integrate, so that they still maintain high activity and function within the bacterial group. This effectively achieves a multi-effect synergy to achieve a comprehensive effect of soil carbon sequestration, pollution reduction and yield increase.

[0077] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A multi-functional synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase, characterized in that: It includes functional microbial communities for degrading organic pollutants, functional microbial communities for soil carbon sequestration, and functional microbial communities for producing small molecule organic matter; the functional microbial communities for degrading organic pollutants include highly efficient pollutant-degrading bacteria; the functional microbial communities for soil carbon sequestration include ammonia-oxidizing bacteria, ammonia-oxidizing archaea, complete ammonia-oxidizing bacteria, sulfate-reducing bacteria, iron-reducing bacteria, rhizobia, and photosynthetic bacteria; the functional microbial communities for producing small molecule organic matter include heterotrophic nitrifying-aerobic denitrifying bacteria, bacterial flocs, polyphosphate-accumulating bacteria, and photosynthetic bacteria.

2. The multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase according to claim 1, characterized in that: The raw materials include the following parts by weight: 5-8 parts ammonia-oxidizing bacteria, 5-8 parts ammonia-oxidizing archaea, 5-8 parts complete ammonia-oxidizing bacteria, 2-5 parts sulfate-reducing bacteria, 2-6 parts iron-reducing bacteria, 5-11 parts rhizobia, 22-30 parts highly efficient pollutant-degrading bacteria, 20-25 parts heterotrophic nitrification-aerobic denitrification bacteria, 8-14 parts bacterial flocs, 3-6 parts polyphosphate-accumulating bacteria, and 6-12 parts photosynthetic bacteria.

3. The multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase according to claim 2, characterized in that: The following raw materials are preferred in parts by weight: 6 parts ammonia-oxidizing bacteria, 6 parts ammonia-oxidizing archaea, 6 parts complete ammonia-oxidizing bacteria, 4 parts sulfate-reducing bacteria, 3 parts iron-reducing bacteria, 6 parts rhizobia, 25 parts highly efficient pollutant-degrading bacteria, 22 parts heterotrophic nitrification-aerobic denitrification bacteria, 10 parts bacterial flocs, 4 parts polyphosphate-accumulating bacteria, and 8 parts photosynthetic bacteria.

4. The multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase according to claim 1, characterized in that: The ammonia-oxidizing bacteria include Nitrosospira_multiformis , Nitrosococcus_oceani , Nitrosomonas_ europaea One or more combinations of the following; ammonia-oxidizing archaea include Nitrosopumilus_maritimus and / or Nitrososphaera_gargensis Complete ammonia-oxidizing bacteria include Candidatus_Nitrospiranitrosa and / or Candidatus_Nitrospiranitrificans Sulfate-reducing bacteria include Pseudomonas_sp. HLS-6 and / or Bacillus cereus Iron-reducing bacteria include Shewanella_oneidensis , Geobacter_sp. , Acidimicrobium One or more combinations of the following; rhizobia include Agrobacterium , Azorhizobium , Bradyrhizobium , Devosia_geojensis One or more combinations thereof.

5. The multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase according to claim 1, characterized in that: The photosynthetic bacteria include Rhodopseudomonas_palustris and / or Rhodopseudomonas faecalis .

6. The multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase according to claim 1, characterized in that: The highly efficient pollutant-degrading bacteria include Enterobacter_cloacae , Saccharomonospora azurea , Nocardiopsis_ alba , Sphingomomonas_yanoikuyae One or more combinations thereof.

7. The multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase according to claim 1, characterized in that: The heterotrophic nitrifying-aerobic denitrifying bacteria include Acinetobacter_baumannii , Alcaligenes faecalis , Paracoccus denitrificans , Microbacterium_testaceum , Thiobacillus_denitrificans One or more combinations of the following; fungal micelles include Zoogloea_oryzae and / or Mitsuaria_chitosanitabida Polyphosphate-accumulating bacteria include Tetrasphaera_elongata and / or Paenibacillus lautus CL-5 .

8. A method for preparing a multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase, characterized in that: Preparing the raw materials for the synergistic microbial agent according to any one of claims 1 to 7 includes the following steps: Step 1: Based on the organic pollutant degradation functional bacteria, add heterotrophic nitrifying-aerobic denitrifying bacteria, bacterial flocs, and rhizobia in sequence according to the proportion, mix well and record it as mixture number one; Step 2: Mix sulfate-reducing bacteria and iron-reducing bacteria in a certain proportion, then add ammonia-oxidizing bacteria, ammonia-oxidizing archaea, and complete ammonia-oxidizing bacteria in sequence. Mix well and let stand for 30-60 minutes. This mixture is called Mixture No.

2. Step 3: Mix Mixture 1 and Mixture 2 1-2 hours before use, add photosynthetic bacteria, place under natural light, and let stand still to obtain synergistic bacterial agent.

9. The application of the multi-effect synergistic microbial agent for soil carbon sequestration, pollution reduction, and yield increase according to any one of claims 1 to 7, or the synergistic microbial agent prepared by the method of claim 8, in improving arable land capacity, characterized in that: The following application methods are included: Before application, the soil is loosened to control the total porosity of the soil to 45-65%, the aeration porosity to >10%, and the soil moisture content to >15%; then the synergistic microbial agent prepared within 5 hours is applied to the soil surface and then tilled to the soil layer within 20cm.

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