A fertilization method for carbon sequestration and emission reduction of wheat-corn rotation soil based on microalgae biofertilizer and application thereof

Abstract

This invention relates to the field of agricultural planting technology, specifically to a fertilization method and its application based on microalgae bio-fertilizer for soil carbon sequestration and emission reduction in wheat-corn rotation. The fertilization method for soil carbon sequestration and emission reduction in wheat-corn rotation includes, in a wheat-corn rotation system, after harvesting corn and wheat, crushing and returning corn and wheat straw to the field; spraying microalgae bio-fertilizer that promotes soil carbon sequestration and emission reduction on the soil surface; and after sowing wheat and corn, applying soil conditioner and compound fertilizer. Based on a winter wheat-summer corn rotation system, this invention, focusing on the goals of carbon sequestration, emission reduction, stable yield, and increased efficiency, combines the application of microalgae bio-fertilizer to establish a fertilization and tillage method that enhances the carbon sequestration capacity of arable land and the level of green production. This method improves fertilizer utilization at the source and reduces the greenhouse gas emission intensity of the entire production system, possessing significant promotional value.

Description

Technical Field

[0001] This invention relates to the field of agricultural planting technology, specifically to a fertilization method based on microalgae bio-fertilizer, applicable to soil carbon sequestration and emission reduction in wheat-corn rotation, and its application. Background Technology

[0002] Global climate change profoundly impacts human survival and development, posing a significant threat to the environment and food security. Atmospheric CO2, CH4, and N2O are the main contributors to the greenhouse effect, with their increased concentrations accounting for nearly 80% of the overall increase in greenhouse gas emissions. Agricultural activities are a major source of greenhouse gas emissions, and farmland ecosystems are both sources and sinks of these emissions. CH4 and N2O are the most significant agricultural greenhouse gases, with farmland soil being the primary source of N2O emissions. In most soils, the amount and type of nitrogen fertilizer applied are the most important factors influencing N2O emissions from farmland.

[0003] Soil serves as the foundation for the stable development of terrestrial ecosystems, and its carbon pool accounts for over 60% of the total terrestrial ecosystem carbon pool. Soil carbon includes soil organic carbon (SOC) and soil inorganic carbon, with SOC being the most active and susceptible to changes in the global carbon pool due to agricultural management practices. Soil carbon sequestration refers to the process of increasing the content of organic and inorganic carbon in soil through improved human management practices, thereby fixing atmospheric CO2 into the soil carbon pool. Increasing soil carbon sequestration not only provides carbon sources for plant growth and microbial reproduction, optimizes soil structure, and promotes the release and transformation of nutrients in the soil, but it is also an effective measure to reduce greenhouse gas emissions into the atmosphere. Therefore, accelerating the reduction and carbon sequestration of agricultural and rural emissions, lowering the intensity of greenhouse gas emissions from agricultural and rural production and daily life, and improving the carbon sequestration capacity of farmland soils are conducive to enhancing the ability of agricultural production to adapt to climate change and making a positive contribution to global climate change mitigation.

[0004] The double crop rotation of summer corn and winter wheat is one of the most important highly intensive grain production systems in northern China. This system produces a huge amount of crop straw, which is rich in nutrients and, when returned to the field, can potentially increase soil fertility and carbon sequestration, thereby improving farmland productivity. However, returning straw alone has certain drawbacks. For example, straw has a high carbon-nitrogen ratio, decomposes relatively slowly, and cannot provide nutrients to crops in a timely manner. Furthermore, in the early stages of straw return, the decomposition process can even lead to competition for nitrogen with crops, affecting crop growth. Therefore, fertilizers should be applied simultaneously with straw return to achieve the dual goals of accelerating straw decomposition and meeting crop growth needs.

[0005] Microalgae are a type of microorganism capable of photosynthesis, fixing CO2 while absorbing large amounts of nutrients and synthesizing various high-value metabolites, including polysaccharides, proteins, carotenoids, unsaturated fatty acids, and various minerals. As a novel type of microbial fertilizer, microalgae bio-fertilizer demonstrates unique value and potential in promoting plant growth, enhancing their stress resistance, and improving soil fertility.

[0006] Chinese patent application CN122123234A discloses a method for stable yield and carbon sequestration through organic fertilizer application in wheat-maize rotation. The method includes: constructing and applying a gradient slow-release organic matrix composed of granular organic fertilizer, microalgae bio-fertilizer, straw, and biochar to establish an initial carbon and nitrogen pool; diagnosing soil carbon activation index and nitrogen environmental risk index at the maize's tasseling stage to quantitatively assess the conversion efficiency and environmental risk of this carbon and nitrogen pool; based on these two indices, synergistically and precisely regulating exogenous carbon input and chemical nitrogen fertilizer reduction in the subsequent wheat season; and iteratively optimizing the matrix ratio and diagnostic control thresholds based on the overall performance of yield, carbon sequestration, and emission reduction in the rotation cycle. However, this method has a complex technical route and a high operational threshold, hindering its large-scale application. Chinese patent application CN117586075A discloses a microalgae mineral organic fertilizer, its preparation method, and its application, belonging to the field of biofertilizer technology. The preparation method involves mixing a composite microalgae nutrient solution containing *Chlorella protozoa*, *Anabaena*, and *Monoflagellates* with modified palygorskite and chicken manure, followed by fermentation to obtain the microalgae mineral organic fertilizer. However, this fertilizer has only been tested for its effectiveness on cabbage crops and is not suitable for application in the complex planting system of summer corn and winter wheat rotation. Chinese patent application CN106342432A discloses a method for promoting soil carbon sequestration in a corn-wheat rotation system. Specifically, after harvesting corn and wheat, the corn and wheat straws are crushed and returned to the field. A microbial compound inoculant that promotes straw fermentation and decomposition is sprayed onto the straw surface, and organic fertilizer is applied. After sowing corn and wheat, a soil conditioner containing nitrogen-fixing bacteria and compound fertilizer are applied, and plant amino acid liquid fertilizer is sprayed during the development period of corn and wheat. However, this method relies on heterotrophic decomposing bacteria to promote straw decomposition for indirect carbon sequestration, and the carbon sequestration effect is limited by straw decomposition efficiency and environmental conditions.

[0007] Therefore, there is an urgent need to develop a microalgae bio-fertilizer and corresponding fertilization method that is suitable for wheat-maize rotation systems, easy to operate, and can achieve synergistic effects of soil carbon sequestration, greenhouse gas emission reduction, and stable crop yield increase. Summary of the Invention

[0008] In order to solve one of the aforementioned technical problems in the existing technology In a first aspect, the present invention provides a fertilization method for soil carbon sequestration and emission reduction in wheat-corn rotation, comprising, in a wheat-corn rotation system, after harvesting corn and wheat, crushing and returning corn stalks and wheat stalks to the field, spraying microalgae bio-fertilizer that promotes soil carbon sequestration and emission reduction on the soil surface, and after sowing wheat and corn, applying soil conditioner and compound fertilizer.

[0009] In some embodiments, the method for preparing the microalgae bio-fertilizer includes the following steps: S1. Algal strain expansion culture: Algae Trichoderma and Chlorella sorokinensis were inoculated into the culture medium and expanded stepwise under suitable temperature and light conditions. S2, Mixing and Encapsulation: Mix the two amplified algal solutions obtained in step S1 at a volume ratio of (1~2):(1~2), add encapsulation agent, and allow algal cells to self-assemble into encapsulated spores. Let it stand in a dark environment. S3. Mesoporous silica encapsulation treatment: The encapsulated spore suspension obtained in step S2 is mixed with the mesoporous silica carrier solution and stirred at a suitable temperature to form a mesoporous shell layer on the surface of the encapsulated spores, thus obtaining a microalgae silica complex. S4. Finished product preparation: Collect the microalgae silica complex obtained in step S3, wash, resuspend, and adjust the microalgae cell concentration, then dispense it into containers to prepare the microalgae bio-fertilizer for soil carbon sequestration and emission reduction in wheat-corn rotation.

[0010] In the optimized active microalgae bio-fertilizer of this invention, the ratio of *Trichophyton variegata* to *Chlorella sorokinense* is (1~2):(1~2), with a preferred ratio of 1:1. This invention combines two different freshwater microalgae in a specific ratio, achieving a high-activity, effective, and long-lasting bio-fertilizer with strong proliferation capacity through a rational formulation.

[0011] In some embodiments, in step S1, the culture temperature is 20~30°C and the light intensity is 2000~4000 lux.

[0012] In some embodiments, in step S1, when the number of upper-level microalgal cells is ≥10 7 When the number of cells / mL reaches a certain level, proceed to the next stage of culture.

[0013] In some embodiments, in step S2, the encapsulating agent is a controlled-release nanoparticle encapsulating agent for tea polyphenols.

[0014] In some embodiments, the tea polyphenol controlled-release nanoparticle encapsulation agent is prepared by an emulsion solvent evaporation method, which includes dissolving polylactic acid in an organic solvent, adding tea polyphenols to form an inner phase, using a gelatin aqueous solution as an outer phase, and then encapsulating the product by stirring, filtering, washing, and drying.

[0015] In some embodiments, the organic solvent is selected from one or more of dichloromethane, ethyl acetate, chloroform, acetone, tetrahydrofuran, nitromethane, acetonitrile, and toluene.

[0016] In some embodiments, the mass ratio of polylactic acid to tea polyphenols is (1~5):(1~5).

[0017] This invention ensures the structural integrity, release characteristics, and effectiveness in inducing cysteine ​​spore formation of microcapsules by controlling the appropriate mass ratio of polylactic acid to tea polyphenols. If the ratio is too high (above 5:1), the release rate of tea polyphenols is too slow, failing to effectively induce algal cell self-assembly to form cysteine ​​spores within a suitable timeframe; if the ratio is too low (below 1:5), it is insufficient to completely encapsulate the tea polyphenols, leading to premature leakage and excessively high local concentrations in the algal solution, causing stress damage to algal cells and affecting the efficiency of cysteine ​​spore formation.

[0018] In some embodiments, the mass fraction of gelatin in the gelatin aqueous solution is 0.5-2.0%.

[0019] In some embodiments, in step S2, the concentration of the encapsulating agent is 0.5 to 1.5 mg / L, and the settling time is 12 to 36 hours.

[0020] In some preferred embodiments, in step S2, the concentration of the encapsulating agent is 1.0 mg / L, and the settling time is 24 hours.

[0021] In some embodiments, in step S3, the volume ratio of the encapsulated spore suspension to the mesoporous silica carrier solution is 1:(2~4).

[0022] In some embodiments, the number of viable microalgae cells in the microalgae bio-fertilizer is ≥1.0 × 10⁻⁶. 8 per mL.

[0023] In some embodiments, the number of viable microalgae cells in the microalgae bio-fertilizer is ≥1.0 × 10⁻⁶. 8 per g.

[0024] In some embodiments, the fertilization method includes pouring the microalgae bio-fertilizer into water and stirring it evenly; the volume ratio of the microalgae bio-fertilizer to water is 1:(250~750) (for example, the microalgae bio-fertilizer is liquid).

[0025] In some embodiments, the fertilization method includes pouring the microalgae bio-fertilizer into water and stirring it evenly; the weight ratio of the microalgae bio-fertilizer to water is 1:(250~750) (for example, the microalgae bio-fertilizer is solid).

[0026] In some embodiments, the fertilization method specifically includes the following steps: (1) Harvesting corn and returning straw to the field: Harvest corn in late September and crush the corn straw to a depth of less than 15cm and return it to the field; (2) Spray microalgae bio-fertilizer on soil surface: dilute microalgae bio-fertilizer with water at a ratio of 1: (250~750) and spray it on the field covered with straw at a rate of 10~15 L / ha. (3) Soil mechanical tillage: After the straw is crushed and evenly covered on the ground, rotary tillage is carried out with a tillage width of more than 1.8m and a tillage depth of 15~18cm. After rotary tillage, the soil is compacted. The root depth qualification rate is ≥85%, the straw burial rate after tillage is ≥70%, and the surface flatness after tillage is ≤5.0cm. (4) Wheat sowing, combined with soil conditioner and compound fertilizer: Sow wheat with a row spacing of 15-30cm, a sowing depth of 2-3cm, and a sowing amount of 135-150kg / ha. After sowing, compact the soil. Mix 300-450kg / ha of compound fertilizer and 150-300kg / ha of soil conditioner evenly and apply them at the same time as sowing. The fertilization depth is 3-5cm below the seed. (5) Conventional field management and topdressing during key growth stages of wheat: During the wheat greening period, apply 150-300 kg / ha of urea; adopt water-saving irrigation technology and irrigate a total of 4 times, watering the roots, overwintering, jointing and heading and flowering in a timely manner; (6) Wheat harvesting and straw return to the field: Wheat is harvested and the straw is crushed to less than 10cm and returned to the field; (7) Spray microalgae bio-fertilizer on the surface of wheat straw: dilute microalgae bio-fertilizer with water at a ratio of 1: (250~750) and spray it on the field covered with straw at a rate of 10~15 L / ha. (8) Direct sowing of summer maize, with the application of soil conditioner and compound fertilizer: Sow maize in fields with a relative moisture content of 65% to 75%, with a sowing rate of 60 to 75 kg / ha and a sowing depth of 3 to 5 cm. Mix 150 to 450 kg / ha of compound fertilizer and 150 to 300 kg / ha of soil conditioner evenly and apply them at the same time when sowing. (9) Conventional field management and foliar fertilizer application during key growth periods of corn: When corn has 10-13 unfolded leaves, add 225-450 kg / ha of urea; water the roots and seeds in time, water the large trumpet mouth and water the flowering and grain filling.

[0027] The composition of the soil conditioner used in this invention is not particularly limited, as long as it improves the physical structure of the soil and enhances its water and fertilizer retention capacity. In some embodiments, the soil conditioner comprises mineral materials, organic materials, and superabsorbent polymers to improve the physical structure of the soil and enhance its water and fertilizer retention capacity; for example, it can be commercially available SoothingSoil conditioner.

[0028] In some embodiments, the compound fertilizer has a nitrogen content of 15% to 18%, a phosphorus pentoxide content of 15% to 21%, and a potassium oxide content of 6% to 15%.

[0029] In some embodiments, the nitrogen fertilizer in the compound fertilizer is urea, and the ratio of N, P2O5 and K2O in the urea is 46:0:0; the phosphate fertilizer in the compound fertilizer is diammonium phosphate, and the ratio of N, P2O5 and K2O in the diammonium phosphate is 18:46:0; and the potassium fertilizer in the compound fertilizer is potassium sulfate, and the ratio of K2O in the potassium sulfate is 0:0:50.

[0030] In some embodiments, the application rate of the compound fertilizer during the wheat season is 180-200 kg N / ha for nitrogen fertilizer, 45-55 kg P / ha for phosphorus fertilizer, and 80-100 kg K / ha for potassium fertilizer.

[0031] In some embodiments, the application rate of the compound fertilizer during the corn season is 140-180 kg N / ha for nitrogen fertilizer, 40-50 kg P / ha for phosphorus fertilizer, and 70-85 kg K / ha for potassium fertilizer.

[0032] Secondly, the present invention provides the application of the fertilization method described in the first aspect in the wheat-maize rotation system for carbon sequestration, emission reduction and yield increase.

[0033] In some embodiments, the carbon sequestration includes an increase in soil organic carbon content, fine particulate organic carbon content, coarse particulate organic carbon content, mineral organic carbon content, soluble organic carbon content, and soil microbial biomass carbon content during the wheat season, relative to conventional fertilization without the application of microalgae biofertilizer.

[0034] In some embodiments, the carbon sequestration includes an increase in soil organic carbon content, fine particulate organic carbon content, coarse particulate organic carbon content, mineral organic carbon content, soluble organic carbon content, and soil microbial biomass carbon content during the maize season, relative to conventional fertilization without the application of microalgae biofertilizer.

[0035] In some implementations, the soil organic carbon content increases by 25% to 35% during the wheat season.

[0036] In some implementations, the content of fine particulate organic carbon in the soil during the wheat season increases by 15% to 50%.

[0037] In some implementations, the coarse particulate organic carbon content in the soil increases by 8% to 30% during the wheat season.

[0038] In some embodiments, the mineral organic carbon content in the soil during the wheat season increases by 120% to 130%.

[0039] In some implementations, the soil organic carbon content increases by 25% to 35% during the maize season.

[0040] In some implementations, the fine particulate organic carbon content in the soil increases by 5% to 10% during the maize season.

[0041] In some implementations, the coarse organic carbon content in the soil during the maize season increases by 50% to 60%.

[0042] In some implementations, the mineral organic carbon content in the soil increases by 1% to 5% during the maize season.

[0043] In some embodiments, the emission reduction includes a reduction in the emission flux of one or more greenhouse gases such as CH4, CO2, and N2O during the wheat or corn season, relative to the absence of the application of the microalgae biofertilizer.

[0044] In some embodiments, the emission reduction includes a reduction in soil N2O emission flux and / or cumulative soil N2O emissions during the wheat or corn season, relative to the absence of the microalgae biofertilizer.

[0045] In some implementations, the soil N2O emission flux during the wheat season is reduced by 46.90%.

[0046] In some implementations, the soil N2O emission flux during the maize season is reduced by 38.20%.

[0047] In some embodiments, the increased yield includes an increase in one or more of the following, relative to conventional fertilization without the application of microalgae biofertilizer: effective panicle number, number of grains per panicle, thousand-grain weight, and biomass.

[0048] In some implementations, the increased yield includes a 35% to 45% increase in maize season biomass.

[0049] This invention is based on the winter wheat-summer maize rotation system. It focuses on the goals of "carbon sequestration, emission reduction, stable yield and increased efficiency". It combines the application of microalgae bio-fertilizer and the technical points of reducing nitrogen application by 20% on the basis of conventional nitrogen application. It clarifies the fertilization and tillage methods to improve the carbon sequestration capacity of arable land and the level of green production.

[0050] Compared with the prior art, the beneficial effects of the present invention are at least as follows: (1) Soil carbon sequestration and soil fertility enhancement.

[0051] It increases the reserves of organic carbon (coarse / fine particulate organic carbon, mineral-bound organic carbon), soluble organic carbon and microbial biomass carbon in various soil components, and has good potential for soil sequestration and quality improvement.

[0052] (2) Greenhouse gas emission reduction.

[0053] Optimizing nitrogen fertilizer management and promoting reduced fertilization have significantly reduced the cumulative N2O emissions from farmland soil and the emission intensity per unit yield. This not only reduces fertilizer production costs and upstream carbon emissions, but also effectively reduces N2O emissions during nitrogen conversion in the field, making it a direct and effective way to achieve agricultural emission reduction.

[0054] (3) Ensure high and stable crop yields.

[0055] It has achieved an increase in the yield of winter wheat and summer maize and its components (number of effective ears, number of grains per ear, and thousand-grain weight), demonstrating good potential for stable yield and increased efficiency.

[0056] (4) Integrate conservation tillage and precision management to build a low-carbon production system.

[0057] This invention deeply integrates the fertilization measures of this invention with conservation tillage systems (such as no-till in the corn season and rotary tillage in the wheat season). At the same time, it combines water and fertilizer integration technology to achieve simultaneous and precise supply of water and fertilizer during the critical period of topdressing. This improves fertilizer utilization at the source, reduces the greenhouse gas emission intensity of the entire production system, and constructs a resource-efficient and environmentally friendly low-carbon agricultural model, which has important promotional value. Attached Figure Description

[0058] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of some embodiments of the present invention or the prior art are briefly introduced below. It should be noted that, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0059] Figure 1 A flowchart of a method for preparing microalgae biofertilizer according to an embodiment of the present invention is shown.

[0060] Figure 2 The results show the soil organic carbon content at the wheat season maturity period.

[0061] Figure 3 The results show the soil organic carbon content at the maize maturity period.

[0062] Figure 4 The results of soil organic carbon content during the wheat season are shown.

[0063] Figure 5 The results of soil organic carbon content at the maize season maturity are shown.

[0064] Figure 6 The results show the soluble organic carbon content in the soil at wheat maturity.

[0065] Figure 7The results show the soil microbial biomass carbon content during the wheat grain-filling stage.

[0066] Figure 8 The results of soil microbial biomass carbon content during the maize jointing stage are shown. Detailed Implementation

[0067] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. It should also be understood that the terminology used in the embodiments of this invention is for describing specific implementation schemes and not for limiting the scope of protection of this invention.

[0068] definition Unless otherwise defined, all technical terms, symbols, and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0069] In the description of the invention, the numerical values ​​of time, temperature, ratio, and mass involved can be based on actual measurements, standard equipment parameters, simplified rounding results, or within an acceptable error range, ensuring the practicality and repeatability of the invention.

[0070] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references.

[0071] The terms “about” or “around” as used herein are as understood by one of ordinary skill in the art and vary within a range depending on the context in which they are used. If one of ordinary skill in the art is not familiar with the use of the term in the context in which it is used, “about” or “around” will mean a particular value plus or minus 10%, such as 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0072] Unless otherwise specified, when microalgae content, fertilizer application rate, seeding rate, or other values ​​or parameters used herein are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, they should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1~5” is disclosed, the described range should be interpreted as including ranges “1~4”, “1~3”, “1~2”, “1~2 and 4~5”, “1~3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range includes its endpoints and all integers and fractions within that range.

[0073] The terms “comprising” and / or “including” as used herein specify the presence of the stated features, integers, steps, operations, units and / or components, without excluding the presence or addition of one or more other features, integers, steps, operations, units, components and / or combinations thereof.

[0074] The "microalgae" used in this article refer to a wide variety of photosynthetic autotrophic microorganisms widely distributed on Earth. These microscopic and simple-structured microorganisms play a crucial role in energy conversion and carbon cycling. The "microalgae bio-fertilizer" used in this article is a microbial fertilizer prepared from microalgae, its main components including prokaryotic cyanobacteria and eukaryotic green algae cells. Compared to chemical fertilizers, bio-fertilizers derived from microalgae can effectively improve the utilization rate of nitrogen, phosphorus, and potassium in the soil, enhance crop resistance to stress, promote plant growth, improve the quality and yield of agricultural products, and significantly reduce the toxicity of plant pathogens in the soil. Algal cells possess nitrogen-fixing capabilities, which can continuously increase the nutrient content in the soil and enhance the crop's resistance to adversity by regulating the structure of the soil microbial community; simultaneously, biological nitrogen fixation constitutes the main source of nitrogen in natural ecosystems. Nitrogen-fixing cyanobacteria use heterocellular structures to convert atmospheric molecular nitrogen into nitrogen compounds to meet the needs of plants and animals in the soil. In terms of the preparation form of algal fertilizer, it mainly exists in liquid form. In some embodiments, the microalgae bio-fertilizer uses whole algal active cells as a substrate. Its advantages lie in the fact that the microalgae cells can survive and reproduce, and are free of algal toxins, exhibiting high safety, meeting ecological and environmental standards, and being easily absorbed by plants. Live microalgae biomass can reduce carbon dioxide emissions through atmospheric carbon capture and sequestration, thus making a significant contribution to the development of green and sustainable agriculture. In some embodiments, the active microalgae biofertilizer of the present invention comprises two types of live microalgae cells, namely, *Trichophyton spp.* (…). Trichormus variabilis ) and Sorokin Chlorella ( Chlorellasorokiniana The variable Trichophyton spp. has strong nitrogen fixation, reproductive capacity, and extracellular polysaccharide secretion capacity. Chlorella sorokinense has strong chlorophyll content, protein content, and extracellular polysaccharide secretion capacity.

[0075] Variegated Trifoliolus Trichormus variabilis Formerly known as the variable fish algae Anabaena variabilis It belongs to the phylum Cyanobacteria, class Cyanobacteria, order Nostocales, family Nostocaceae, and genus *Trichophyta*. In some embodiments, the variable *Trichophyta* is selected from any one or more of the following: *Trichophyta 0441*, *Trichophyta ATCC29413*, *Trichophyta FACHB-164*, *Trichophyta FACHB-171*, *Trichophyta* genus FACHB-319, *Trichophyta SAG 1403-4b*, *Trichophyta A2*, *Trichophyta 9RC*, *Trichophyta N2B*, *Trichophyta PNB*, *Trichophyta ARAD*, *Trichophyta FSR*, *Trichophyta V5*, *Trichophyta NIES-23*, and *Trichophyta str. HINDAK 2001 / 4*. In some specific embodiments, the variable *Trichophyta* is *Trichophyta* genus FACHB-319.

[0076] Sorokin Chlorella Chlorellasorokiniana It belongs to the phylum Chlorophyta, subphylum Chlorophyta, class Chlorophyta, order Chlorellales, family Chlorellaceae, and genus Chlorella. In some embodiments, the *Chlorella sorokinense* is selected from any one or more of the following: *Chlorella sorokinense* ATCC22521, *Chlorella sorokinense* 1230, *Chlorella sorokinense* 1602, *Chlorella sorokinense* 1228, *Chlorella sorokinense* 1412, *Chlorella sorokinense* SLA-04, *Chlorella sorokinense* DSCG149, *Chlorella sorokinense* DSCG147, *Chlorella sorokinense* DSCG150, *Chlorella sorokinense* UTEX1663, and *Chlorella sorokinense* CNDG001. In some specific embodiments, the *Chlorella sorokinense* is *Chlorella sorokinense* 1230.

[0077] Soil organic carbon (SOC), as used in this paper, is an important component of the global carbon cycle and also an indicator of soil fertility. It not only provides nutrients and a suitable environment for plant growth but also provides an energy source for microbial growth and development, making it crucial for soil productivity and the global carbon cycle. Soil organic carbon is a highly complex and continuous mixture containing various organic carbon components and forms. The properties of each component are not entirely the same, and their content in the soil organic carbon pool affects soil carbon transformation, thereby influencing changes in soil quality and the carbon cycle. Based on the determination of organic carbon content, this paper measures the content of different components, including coarse-grained organic carbon, fine-grained organic carbon, mineral-bound organic carbon, soluble organic carbon, and microbial biomass carbon. This invention uses the high-temperature external heating potassium dichromate oxidation-volume method to determine soil organic carbon content, with specific steps referring to the method described in *Soil Agricultural Chemical Analysis* (edited by Lu Rukun, China Agricultural Science and Technology Press, 2000).

[0078] The term "particulate organic carbon" used in this paper refers to the organic carbon portion bound to sand particles, primarily derived from the decomposition products of plant residues with moderate decomposition rates, and organically combined with soil aggregates. Particulate organic carbon is a transitional component in the transformation of fresh organic matter into humus, belonging to a relatively easily decomposed and highly bioactive component of the soil organic carbon pool; this portion of organic carbon is also considered the non-protected part of organic carbon. Based on particle size, it can be divided into coarse particulate organic carbon (>250 μm) and fine particulate organic carbon (53~250 μm), with particles <53 μm classified as mineral-bound organic carbon. The recalcitrant portions of plant residues, after physical crushing, weathering, and degradation, ultimately form coarse particulate organic carbon. During flooding, the easily degradable portions of plants can be utilized by microorganisms to synthesize new organisms, and the dead microorganisms eventually enter the soil, forming fine particulate organic carbon. This invention uses the high-temperature external heating potassium dichromate oxidation-volume method to determine the soil organic carbon content of each component, obtaining the contents of coarse particulate organic carbon (cPOC), fine particulate organic carbon (fPOC), and mineral-bound organic carbon (MAOC). The specific steps are described in "Soil Agricultural Chemical Analysis" (edited by Lu Rukun, China Agricultural Science and Technology Press, 2000).

[0079] The term "mineral-bound organic carbon" used in this article refers to dissolved organic carbon from plant sources that is directly adsorbed onto the mineral surface (external), or formed through microbial-mediated transformation and the binding of residual substances with minerals (internal). Due to its relatively high content and slow and stable turnover rate in soil, it is considered soil-stabilized organic carbon. From a large landscape and time scale perspective, significant changes in soil organic carbon transformation and storage may be attributed to changes in mineral-bound organic carbon.

[0080] The term "dissolved organic carbon (DOC)" as used in this article refers to the portion of organic carbon that is soluble in water or dilute salt solutions. As the most active organic carbon component in soil, soluble organic carbon is the main form of organic carbon migration and transformation. It is usually decomposed and utilized by microorganisms, and then transformed into more stable organic carbon stored in the soil, or degraded and released into the atmosphere in the form of CO2. Soluble organic carbon accounts for a small proportion of the total organic carbon in soil, but it is the main energy source for microbial growth and can serve as an important indicator of microbial growth and readily available nutrient resources for biological decomposition. This invention uses a TOC analyzer to determine the content of soluble organic carbon in soil. Soluble organic carbon in soil is extracted with deionized water, and the concentration of organic carbon in the filtrate is determined by a TOC analyzer. The specific steps are in accordance with the method described by Jones and Willett (Experimental evaluation of methods to quantify dissolved organic nitrogen (DON) and dissolved organic carbon (DOC) in soil, Soil Biology and Biochemistry, Volume 38, Issue 5, 2006 38(5): 991-999).

[0081] The term "microbial biomass carbon (MBC)" used in this article refers to microbial biomass carbon in soil with a volume of 5–10 μm³. 3 The organic carbon contained in living microorganisms is the most active and variable part of soil organic matter. Soil microbial biomass carbon accounts for a relatively small proportion of total soil organic carbon, generally only 0.3%–7%. However, as direct participants and decomposers in soil metabolism, microorganisms are an indispensable part of the soil and are sensitive to agricultural practices, thus serving as active organic carbon. Compared to total soil organic carbon, microbial biomass carbon responds more quickly to changes in soil management practices such as tillage and straw cultivation, and can serve as an early indicator of changes in total soil organic carbon. The ratio of microbial biomass carbon to total soil organic carbon is an important indicator of soil microbial carbon availability, carbon loss, and soil carbon stability. In this invention, the microbial biomass carbon content is determined using the chloroform fumigation and K2SO4 extraction method. The specific steps are as described in GB / T39228-2020 "Determination of Soil Microbial Biomass - Fumigation Extraction Method".

[0082] The term "greenhouse gas" as used in this paper refers to gaseous components in the atmosphere that are naturally occurring or produced by human activities, capable of absorbing and emitting radiation with wavelengths in the infrared spectrum from the Earth's surface, atmosphere, and clouds. These include carbon dioxide (CO2), methane (CH4), nitrous oxide (N2O), hydrofluorocarbons (HFCs), perfluorocarbons (PFCs), sulfur hexafluoride (SF6), and nitrogen trifluoride (NF3). Global warming caused by the increase of greenhouse gases such as methane, nitrous oxide, and carbon dioxide poses a significant challenge to ecological sustainability and food security. Agricultural production activities are one of the most significant sources of greenhouse gas emissions; therefore, reducing greenhouse gas emissions from agricultural production is crucial for mitigating the greenhouse effect and ensuring ecological sustainability and food security. This invention employs a static dark chamber-gas chromatography method to determine greenhouse gas emissions from farmland, using a gas chromatograph (Agilent 7890B) to measure greenhouse gas concentrations.

[0083] As used in this article, crop “yield” refers to the amount (e.g., determined by weight, volume, processed product, or size) or number of tissues or organs produced per plant or per growing season. Therefore, increased yield may affect the economic benefits that can be obtained from a crop within a given growing region and / or growing period. It should be noted that crop yield can be affected by a variety of parameters, including but not limited to: crop biomass; crop vigor, stress tolerance, growth rate; seed yield; number of seeds or grains; seed or grain quality; oil, starch, and / or protein content in harvestable organs (e.g., crop seeds or vegetative organs); number of florets per spike (expressed as the ratio of full seeds to primary spikes); harvest index; number of crops planted per unit area; number and size of harvestable organs per plant and per unit area; number of crops per plant area (density); total number of harvestable organs in the field; total leaf area; carbon assimilation and carbon distribution (distribution / allocation of carbon within the crop); shade tolerance; number of harvestable organs (e.g., seeds), number of seeds per pod, weight of a single seed; and structural improvements (e.g., increasing stem diameter, thickness, or improving physical properties such as elasticity). In some embodiments, the yield parameters described herein include, but are not limited to, spike length, number of spikes, number of grains per spike, thousand-grain weight, yield, and seed setting rate.

[0084] The term "crop biomass" as used in this article refers to the total amount of tissue produced by a crop during a growing season (e.g., measured in grams of air-dried tissue), which can determine or influence crop yield or yield per unit area. Increases in crop biomass can be observed in the whole crop or parts thereof, such as the above-ground (harvestable) parts, vegetative biomass, roots, and seeds. This invention uses the oven-drying gravimetric method to determine crop biomass; the specific steps are described in *General Crop Cultivation* (edited by Cao Weixing, Science Press, 2017).

[0085] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0086] Example Unless otherwise stated, the present invention will be carried out using conventional techniques from botany, biology, etc., which are described in the literature or performed according to product instructions within the scope of this art. Unless otherwise specified, the materials, reagents, or instruments used in the examples are all commercially available conventional products. All quantitative experiments in the following examples were performed in at least three replicates, and the results were averaged.

[0087] like Figure 1 As shown, in one embodiment of the present invention, a method for preparing microalgae bio-fertilizer is provided, comprising the following steps: S1, Algal strain amplification culture *Trichophyton variegata* and *Chlorella sorokinense* were separately inoculated into the culture medium, with temperature, light intensity, and pH controlled. The culture was then expanded stepwise until the number of cells from the previous stage was ≥10. 7 When the number of cells / mL reaches a certain level, proceed to the next stage of culture; S2, Microalgae Mixing and Encapsulation Treatment After mixing the two amplified algal solutions obtained in step S1 at a volume ratio of (1~2):(1~2), 1 mg / L of encapsulation agent was added using a gradient addition method. The algal cells were then stirred magnetically (200 rpm) to form encapsulated spores, and the mixture was left to stand in a dark environment (0 lux) for 24 hours. The encapsulation agent is a controlled-release nanoparticle encapsulation agent for tea polyphenols. The encapsulation agent is prepared by an emulsion solvent evaporation method, which includes dissolving polylactic acid in an organic solvent, adding tea polyphenols to form an inner phase, with the mass ratio of polylactic acid to tea polyphenols being (1~5):(1~5), using a 0.5%~2.0% gelatin aqueous solution as the outer phase, and obtaining the encapsulation agent through emulsification, stirring, filtration, washing, and drying.

[0088] S3, mesoporous silica encapsulation treatment The cyst spore suspension obtained in step S2 was mixed with the mesoporous silica carrier solution at a volume ratio of 1:(2~4), and stirred at a suitable temperature to form a mesoporous shell layer on the surface of the cyst spores, thus obtaining a microalgae silica complex.

[0089] S4. Finished Product Preparation The microalgal silica complex obtained in step S3 was collected, washed, resuspended, and the algal cell concentration was adjusted to be no less than 1.0 × 10⁻⁶. 8After dividing the microalgae into mL, it is dispensed into containers to obtain the finished microalgae bio-fertilizer.

[0090] The microalgal bio-fertilizer prepared by this invention is an algal active cell liquid composed of *Trichophyton variegata* and *Chlorella sorokinense*, with a single-cell algal content exceeding 1.0 × 10⁻⁶ cells per mL. 8 The algal solution has a pH of 6.5-7.5, a total carbon concentration of 300-400 mg / L, a total nitrogen concentration of 450-550 mg / L, a total phosphorus concentration of 10-15 mg / L, and a chlorophyll content of 3-5 mg / L. The microalgae in this invention's microalgae bio-fertilizer are in a living state, with a shelf life exceeding 12 months.

[0091] Example 1: Preparation of Microalgae Biofertilizer The microalgae bio-fertilizer of this invention uses commercially available polymorphic trifoliolone (Pterocarya spp.) Trichormus variabilis FACHB-319 and Chlorella sorokinosa ( Chlorella sorokiniana )1230, the nitrogen-fixing ability, reproductive ability and extracellular polysaccharide secretion ability of Trichophyton moniliforme are strong; Chlorella sorokinensis has high chlorophyll content, protein content and strong extracellular polysaccharide secretion ability.

[0092] The specific steps for preparing the microalgae bio-fertilizer of the present invention are as follows: 1) Select algal species with a single-celled number ≥ 10 7 Algal strains were transferred at a ratio of 1:10 with sterilized BG11 medium, and then transferred in increments of 100 mL, 1 L, 5 L, and 18 L. The transferred algal strains were cultured at a temperature of 25-28℃ under continuous light (light intensity of 2000-4000 lux).

[0093] 2) Monitor algal cell concentration daily using an algal concentration monitor. When the microalgal cell concentration reaches the target concentration value (≥10), 7 Stop culturing ( / mL). Mix the amplified *Trichophyton spp.* and *Chlorella vulgaris* spp. at a 1:1 volume ratio and place in a photoreactor.

[0094] A gradient dosing method was used to inject controlled-release microparticle encapsulation agents (1 mg / L) into the photoreactor, and nanoscale dispersion was achieved using a magnetic stirring system (stirring speed of 200 rpm). Specifically, the encapsulation agent was divided into three equal portions, and one portion was added every 30 minutes.

[0095] 3) Under constant temperature conditions of 25℃~28℃, algal cells self-assemble to form encapsulated spores, with a polymerization efficiency of 1 spore / 100 cells. After being transferred to a completely dark environment (0 lux) and allowed to stand for 24 hours, the encapsulated spores were functionalized and passivated using mesoporous silica encapsulation controlled-release technology. Specifically, the encapsulated spore suspension was added to a mesoporous silica carrier at a volume ratio of 1:2, and the mixture was stirred at 200-300 rpm for 4-6 hours at room temperature (20~30℃) to allow silica to polymerize in situ on the surface of the encapsulated spores, forming a mesoporous shell. The precipitate was then collected by centrifugation and washed three times with deionized water to obtain the passivated algal-silica composite.

[0096] 4) Resuspend the washed precipitate in sterile deionized water and adjust the algal cell concentration to 1.0 × 10⁻⁶. 8 Cells / mL. The passivated algae and controlled-release materials form a stable composite system, which is then packaged in a 200mL high-density polyethylene (HDPE) plastic container to prepare a microalgae-based bio-fertilizer with slow-release function.

[0097] The microalgae bio-fertilizer prepared in this embodiment is a liquid containing active algal cells, composed of *Trichophyton variegata* and *Chlorella sorokinense*, with a single-cell algal content of 1.0 × 10⁻⁶ cells per mL. 8 The algal solution had a pH of 7.09, a total carbon concentration of 359.70 mg / L, a total nitrogen concentration of 505.55 mg / L, a total phosphorus concentration of 12.39 mg / L, and a chlorophyll content of 4.12 mg / L.

[0098] The microalgae bio-fertilizer of this invention has a shelf life of over 12 months. The microalgae are in a living state, ensuring long-lasting effects. The formula is rationally proportioned, and the two types of microalgae, when combined, exhibit excellent symbiotic proliferation characteristics. Cell counting chamber analysis shows that the number of viable microalgae cells exceeds 1.0 × 10⁻⁶. 8 The cell count / mL and chlorophyll content experiments showed that the cells had high activity and vigorous vitality.

[0099] Example 2 Field Application This embodiment uses the microalgae bio-fertilizer (10) prepared in Example 1. 8 (1 algal cell / mL) Under the straw return technology system, which is currently widely promoted in high-yield grain fields of winter wheat-summer maize in northwestern Shandong, combined with the application of microalgae bio-fertilizers, emission reduction and efficiency improvement and soil carbon sequestration are achieved.

[0100] 2.1 Test Site The Modern Agricultural Science and Technology Park in Dezhou City, Shandong Province, is located at 116°34.2′ longitude and 37°35.4′ latitude. It cultivates two crops a year, primarily winter wheat and summer maize in rotation. The soil is fertile, sandy, and slightly alkaline. For a long time, it has adopted conservation tillage measures, such as returning crushed wheat straw to the field after rotary tillage and then sowing, and no-till sowing with straw mulch returned to the field during the maize season.

[0101] 2.2 Test Materials The summer maize variety is "Denghai 605," sown in mid-June and harvested in mid-October, with a planting density of 26 kg / ha, row spacing of 60 cm, and plant spacing of 25 cm. Straw is returned to the field before sowing, and no-till tillage is the primary method of land preparation. The winter wheat variety is "Jimai 22," sown in late October and harvested in early June. Wheat is sown in rows at a seed rate of 260 kg / ha, with a row spacing of 20 cm. Straw is returned to the field before sowing, and rotary tillage is the primary method of land preparation, with deep plowing once every three years.

[0102] 2.3 Methods The microalgae bio-fertilizer bottled liquid obtained in Example 1 (10 8 The application rate is 12 L / ha (1 algal cells / mL), diluted with water at a ratio of 1:500, and sprayed onto the soil surface using a sprayer. Combined with straw return to the field, it effectively enhances soil microbial activity, promotes nutrient transformation, improves soil structure, and promotes soil carbon sequestration.

[0103] The operational process includes: 1) Harvesting corn, with all corn stalks crushed and returned to the field; 2) Spraying microalgae bio-fertilizer onto the surface of the stalks; 3) Mechanical tillage of the soil (rotary tillage or deep tillage); 4) Wheat sowing, with the application of soil conditioner and compound fertilizer; 5) Topdressing, irrigation, and conventional field management; 6) Harvesting wheat while simultaneously crushing and returning all wheat stalks to the field; 7) Spraying microalgae bio-fertilizer onto the surface of the stalks; 8) No-till direct seeding of summer corn, with the application of soil structure conditioner and compound fertilizer; 9) Topdressing, irrigation, and field management. The fertilizer application schemes for the wheat and corn seasons are shown in Table 1. Urea contains 46% N, diammonium phosphate contains 18% N and 46% P2O5, and potassium sulfate contains 50% K2O. The 20% reduction in nitrogen indicates that, compared to conventional fertilization, the application rates of urea, diammonium phosphate, and diammonium phosphate are all reduced by 20%.

[0104] 2.3.1 Wheat Season Operations (1) Corn harvesting and straw return to the field In late September, corn is harvested using a combine harvester. The corn stalks are then shredded to a thickness of less than 15cm and returned to the field using a corn stalk shredder. Alternatively, the corn can be manually harvested and then the stalks returned to the field using a stalk shredder. The stalk shredding qualification rate should be ≥90%, the missed shredding rate ≤1.5%, the stubble height ≤80mm, the stubble depth ≥50mm, and the stubble shredding qualification rate ≥95%.

[0105] (2) Spray microalgae bio-fertilizer Bottle the microalgae bio-fertilizer liquid (10 8 The application rate is 12 L / ha (1 algal cell / mL), diluted with water at a ratio of 1:500, and then sprayed onto the soil surface using a sprayer.

[0106] (3) Soil mechanical tillage After the straw is crushed and evenly covered on the ground, select a rotary tiller with a tillage width of 1.8m or more and a single beam with intermediate drive. The tillage depth should be 15-18cm. After rotary tillage, compaction is required. The root depth qualification rate should be ≥85%, the straw burial rate after tillage should be ≥70%, and the surface flatness after tillage should be ≤5.0cm.

[0107] (4) Wheat seed treatment, sowing and application of base fertilizer No special treatment is required for the seeds. Seed dressing agents can be used to dress the seeds directly. After drying, the seeds can be sown.

[0108] For fields where straw has been returned to the soil, wheat should be sown in small quantities with a row spacing of 15-30 cm, a sowing depth of 2-3 cm, and a sowing rate of 9-10 kg / mu. After sowing, the soil should be compacted. Sowing time is from October 5th to 10th, and the sowing rate deviation should be controlled within 5%. Seeds should be evenly distributed, sown at a consistent depth, and the sowing depth qualification rate should be ≥80%.

[0109] Mix 20-30 kg / mu of base fertilizer (the base fertilizer should be a compound fertilizer with a nitrogen-phosphorus-potassium ratio of 15-15-15 or a suitable formula fertilizer, which is 1 / 3 to 1 / 2 less than the traditional amount) and 15 kg / mu of soil conditioner evenly.

[0110] The seeds and the well-mixed compound fertilizer and soil conditioner are loaded separately into the seed and fertilizer bins of the seeder for sowing. The fertilizer is applied at a depth of about 4 cm below the seeds to avoid direct contact between the seeds and fertilizer.

[0111] (5) Topdressing Before the wheat turns green and is watered around the Qingming Festival (late March to early April), apply 15 kg / mu of urea as a top dressing (the amount of urea can be reduced by 30-50% depending on the growth).

[0112] (6) Irrigation, weeding and pest and disease control Water-saving irrigation technology was adopted, and irrigation was carried out four times in total, including timely watering of the roots, overwintering, jointing, and heading and flowering. In special circumstances, such as drought in the later stage, an additional watering can be carried out for grain filling.

[0113] (7) Weeding and pest and disease control Herbicides and insecticides can be used as appropriate depending on the severity of the pest infestation, and it is recommended to reduce pesticide use by 50% or more.

[0114] 2.3.2 Corn Season Operations (1) Wheat harvesting and straw return to the field Wheat is harvested using a wheat combine harvester with straw chopping and spreading functions, completing wheat harvesting and straw crushing and returning to the field in one go. The straw chopping length is ≤10cm, the qualified rate of chopping length is ≥90%, the uneven spreading rate is ≤20%, and the missed chopping rate is ≤1.5%.

[0115] (2) Spray microalgae bio-fertilizer The application rate of microalgae bio-fertilizer is 12L / ha. After diluting it with water at a ratio of 1:500, spray it evenly on the field where straw has been laid.

[0116] (3) Corn seed treatment, sowing, basal fertilizer and seedling establishment No special treatment is required for the seeds. Seed dressing agents can be used to dress the seeds directly. After drying, the seeds can be sown.

[0117] A corn direct seeding machine is used to complete stubble clearing, furrowing, weed control, deep application of base fertilizer, and soil compaction in one operation. At sowing time, the relative soil moisture content should be around 70%. If soil moisture is poor, irrigation can be carried out before irrigation in irrigated areas. The seeding rate is 4-5 kg / mu, and the sowing depth depends on soil moisture, ideally 3-5 cm. Base fertilizer should be a 15-15-15 NPK compound fertilizer or a suitable formulated fertilizer, applied at a rate of 20 kg / mu (one-third less than traditional application). The base fertilizer and 15 kg / mu of soil conditioner should be mixed evenly and applied at sowing.

[0118] Thinning should be done in mid-to-late June when seedlings have 4-5 leaves, ensuring uniform seedling density. For gaps within 1 meter, leave two seedlings. For gaps exceeding 1 meter, replant with germinated seeds of the same variety, or transplant with soil attached. The first thinning should be done when seedlings have 3-4 leaves, leaving slightly more seedlings depending on the variety. Subsequent thinning should be done in stages to improve uniformity in the cornfield.

[0119] (4) Topdressing In mid-to-late July, when the corn has 10-13 fully expanded leaves (small trumpet stage), apply an additional 30 kg / mu of urea per mu in conjunction with irrigation (this amount can be reduced depending on the growth).

[0120] (5) Irrigation Irrigate corn according to rainfall. Water the roots and seeds promptly after direct seeding to ensure complete emergence. In late July, during the tasseling stage of summer corn, water thoroughly to meet its growth needs. In mid-August, after fertilization, corn reaches full maturity through the grain-filling, milk-ripe, and wax-ripe stages; at this time, water appropriately during the flowering and grain-filling stages.

[0121] (6) Weeding and pest and disease control Herbicides and insecticides can be used as appropriate depending on the severity of the pest infestation, and it is recommended to reduce pesticide use by 50% or more.

[0122] Table 1 Fertilizer application rates for wheat and corn seasons Note: A 20% reduction in nitrogen means that the application rates of urea, diammonium phosphate, and potassium sulfate have all been reduced by 20% compared to conventional fertilization.

[0123] Example 3 The only difference from Example 1 is that in step 2), the amplified polymorphic trichophyton lysate and the Sorokin microphylla lysate are mixed at a volume ratio of 1:2 and placed in a photoreactor. All other operating steps are the same as in Example 1.

[0124] Example 4 The only difference from Example 1 is that in step 2), the amplified polymorphic trichophyton lysate and the Sorokin microphylla lysate are mixed at a volume ratio of 2:1 and placed in a photoreactor. All other operating steps are the same as in Example 1.

[0125] Example 5 The only difference from Example 1 is that in step 3), the encapsulated spore suspension is added to the mesoporous silica carrier solution at a volume ratio of 1:4. All other operating steps are the same as in Example 1.

[0126] The volume ratio of cyst spore suspension to mesoporous silica carrier solution affects the encapsulation effect of the silica shell. When the proportion of mesoporous silica carrier solution is too low (e.g., greater than 1:2), the silica supply is insufficient, and a continuous and complete mesoporous shell cannot be formed on the surface of all cyst spores. Some spores are not fully encapsulated, affecting the passivation effect and sustained-release performance. When the proportion of mesoporous silica carrier solution is too high (e.g., less than 1:4), the excessive amount of mesoporous silica carrier results in an overly thick shell, which in turn affects the sustained-release effect of the microalgae cells.

[0127] Comparative Example 1 In step 2), the amplified polymorphic trichophyton lysate and the Sorokin microphylla lysate are mixed at a volume ratio of 1:5 and placed in a photoreactor. All other operating steps are the same as in Example 1.

[0128] Comparative Example 2 In step 2), the amplified polymorphic trichophyton lysate and the Sorokin microphylla lysate are mixed at a volume ratio of 5:1 and placed in a photoreactor. All other operating steps are the same as in Example 1.

[0129] The microalgae biofertilizers prepared in Examples 3-5 and Comparative Examples 1-2 were applied according to the same steps described in Method 2.3 of Example 2.

[0130] Example 6 Fertilization was carried out according to the same steps described in method 2.3 of Example 2. The nitrogen content was reduced by 20% and the microalgae bio-fertilizer prepared in Example 1 was applied at a rate of 10 L / ha.

[0131] Example 7 Fertilization was performed following the same steps described in method 2.3 of Example 2. The nitrogen content was reduced by 20% and the microalgae bio-fertilizer prepared in Example 1 was applied at a rate of 15 L / ha.

[0132] Test Example 1: Monitoring of Soil Carbon Sequestration Capacity between Wheat and Maize This invention conducted a field trial of crop rotation across three growing seasons—maize, wheat, and maize—from June 2024 to October 2025. To ensure the comparability of the data and the accuracy of the results, the relevant indicators for the maize season were measured using the results from the second maize season (i.e., the 2025 maize season).

[0133] To verify the effectiveness of this invention, topsoil samples were collected before the start of the experiment, at wheat maturity, and at maize maturity for each treatment. The contents of soil organic carbon, coarse-grained organic carbon, fine-grained organic carbon, mineral-bound organic carbon, soluble organic carbon, and microbial biomass carbon were measured to analyze the improvement effect of this invention on soil carbon sequestration capacity. The analytical methods for each index are as described above; the specific results are as follows.

[0134] 1.1 Soil organic carbon 1) Soil organic carbon content results for each treatment at wheat maturity stage are as follows: Figure 2 As shown in the figure. Treatment 1 represents no fertilization, treatment 2 represents conventional fertilization, and treatment 3 represents nitrogen reduction of 20%. Examples 1 and 3-5 represent nitrogen reduction of 20% combined with microalgae bio-fertilizer prepared in Examples 1 and 3-5; Example 6 represents nitrogen reduction of 20% combined with 10 L / ha of microalgae bio-fertilizer prepared in Example 1; Example 7 represents nitrogen reduction of 20% combined with 15 L / ha of microalgae bio-fertilizer prepared in Example 1; Comparative Examples 1-2 represent nitrogen reduction of 20% combined with microalgae bio-fertilizer prepared in Comparative Examples 1-2 (all the following are the same).

[0135] Depend on Figure 2 It can be seen that, compared with the conventional fertilization treatment in treatment 2, the soil organic carbon content decreased in treatment 1 (no fertilization), treatment 3 (nitrogen reduction of 20%), and treatment 3 (nitrogen reduction of 20%) combined with the microalgae bio-fertilizer prepared in comparison examples 1-2. However, the treatments with nitrogen reduction of 20% combined with the microalgae bio-fertilizers in examples 1, 3-5, and examples 6-7 all showed an improvement effect, with an increase of about 29.88%.

[0136] 2) The soil organic carbon content results for each treatment at the maize maturity stage showed the same trend as those for wheat maturity stage, specifically as follows: Figure 3 As shown. By Figure 3It can be seen that, compared with the conventional fertilization treatment in treatment 2, the soil organic carbon content decreased in treatment 1 (no fertilization), treatment 3 (nitrogen reduction of 20%), and treatment 3 (nitrogen reduction of 20%) combined with the microalgae bio-fertilizer prepared in comparison examples 1-2. However, the treatments with nitrogen reduction of 20% combined with the microalgae bio-fertilizers in examples 1, 3-5, and examples 6-7 all showed an improvement effect, with an increase of about 12.96%.

[0137] 1.2 Soil components: organic carbon 1) The results of coarse organic carbon (coarse), fine organic carbon (fine), and mineral organic carbon (mineral) in the soil of each treatment at wheat maturity are as follows: Figure 4 As shown.

[0138] Depend on Figure 4 It can be seen that, compared with the conventional fertilization treatment in treatment 2, in the 0-10 cm soil layer, the treatment with 20% nitrogen reduction combined with the microalgae bio-fertilizers of Examples 1, 3-5, and the treatments of Examples 6-7 showed a significant increase in fine particulate organic carbon content, with an increase of approximately 46.35% (results of Examples 3-7 are not shown); in the 10-20 cm soil layer, the treatment with 20% nitrogen reduction combined with the microalgae bio-fertilizers of Examples 1, 3-5, and the treatments of Examples 6-7 showed an increase of approximately 26.30% in coarse particulate organic carbon content and approximately 17.42% in fine particulate organic carbon content (results of Examples 3-7 are not shown); in the 20-30 cm soil layer... In the cm soil layer, compared to conventional fertilization, the coarse particulate organic carbon content increased by approximately 9.30% in the treatments with 20% nitrogen reduction combined with the microalgae bio-fertilizers of Examples 1, 3-5, and Examples 6-7 (results for Examples 3-7 are not shown). Simultaneously, the mineral organic carbon content in this soil layer increased by approximately 55.6% in the treatments with 20% nitrogen reduction combined with the microalgae bio-fertilizers of Examples 1, 3-5, and Examples 6-7 (results for Examples 3-7 are not shown). The microalgae bio-fertilizer treatments in Comparative Examples 1-2 did not show a significant increase in soil component organic carbon (results not shown).

[0139] 2) The results of the contents of coarse organic carbon (coarse), fine organic carbon (fine), and mineral organic carbon (mineral) in the soil of each treatment at the maize maturity stage are as follows: Figure 5 As shown.

[0140] Depend on Figure 5It can be seen that, compared with the conventional fertilization treatment in Treatment 2, in the 0-10 cm soil layer, the treatments with 20% nitrogen reduction combined with the microalgae bio-fertilizers of Examples 1, 3-5, and Examples 6-7 showed an increase in fine particulate organic carbon content (results for Examples 3-7 not shown), with an increase of approximately 7.90%. Simultaneously, the increase in mineral organic carbon content in this soil layer was approximately 3.10% (results for Examples 3-7 not shown). In the 20-30 cm soil layer, the increase in coarse particulate organic carbon content was approximately 58.58% (results for Examples 3-7 not shown). The microalgae bio-fertilizer treatments in Comparative Examples 1-2 did not show an improvement effect (results not shown).

[0141] 1.3 Soil soluble organic carbon 1) Regarding the soluble organic carbon content in soil during the wheat season, from the perspective of the entire growth period, the content of soluble organic carbon in soil shows a decreasing trend as the wheat growth period progresses, with the lowest content at maturity. The soluble organic carbon content in soil for each treatment at wheat maturity is as follows: Figure 6 As shown. By Figure 6 It can be seen that, compared with the conventional fertilization treatment in Treatment 2, the treatments with 20% nitrogen reduction combined with the microalgae bio-fertilizers of Examples 1, 3-5, and Examples 6-7 showed a significant increase in soil soluble organic carbon content in all soil layers (results for Examples 3-7 are not shown). The microalgae bio-fertilizer treatments in Comparative Examples 1-2 did not show an increasing effect (results are not shown). At other growth stages, there were no significant differences between the conventional fertilization treatment in Treatment 2 and the treatments with 20% nitrogen reduction combined with the microalgae bio-fertilizers of Examples 1, 3-5, and Examples 6-7.

[0142] 2) Regarding the soluble organic carbon (SOC) content in the soil during the maize season, from the perspective of the entire growth period, the SOC content was highest during the seedling and tasseling stages, and then decreased with the progression of the maize growth period, reaching its lowest level at maturity. Specifically, at maize maturity, the SOC content ranged from 65.85 to 90.99 mg / kg. There was no significant difference between Treatment 2 (conventional fertilization), the treatment with 20% nitrogen reduction combined with the microalgae bio-fertilizers of Examples 1, 3-5, and Examples 6-7. At other growth stages, there were no significant differences between Treatment 2 (conventional fertilization), the treatment with 20% nitrogen reduction combined with the microalgae bio-fertilizers of Examples 1, 3-5, and Examples 6-7.

[0143] 1.4 Soil microbial biomass carbon content 1) Regarding the soil microbial biomass carbon content during the wheat season, the soil microbial biomass carbon content of each treatment during the wheat grain-filling stage is as follows: Figure 7As shown. By Figure 7 It can be seen that, compared with the conventional fertilization treatment in Treatment 2, the soil microbial biomass carbon content of the treatments with 20% nitrogen reduction combined with the microalgae bio-fertilizers of Examples 1, 3-5, and Examples 6-7 was significantly increased in all soil layers (results of Examples 3-7 are not shown). The microalgae bio-fertilizer treatments in Comparative Examples 1-2 did not show an increasing effect (results are not shown). At other growth stages, there were no significant differences between the conventional fertilization treatment in Treatment 2 and the treatments with 20% nitrogen reduction combined with the microalgae bio-fertilizers of Examples 1, 3-5, and Examples 6-7.

[0144] 2) Regarding the soil microbial biomass carbon content during the maize season, the soil microbial biomass carbon content of each treatment at the jointing stage is as follows: Figure 8 As shown. By Figure 8 It can be seen that, compared with the conventional fertilization treatment in Treatment 2, the soil microbial biomass carbon content of the treatments with 20% nitrogen reduction combined with the microalgae bio-fertilizers of Examples 1, 3-5, and Examples 6-7 was increased in all soil layers (results of Examples 3-7 are not shown). A similar increasing effect was also observed during the large trumpet stage. The microalgae bio-fertilizer treatments in Comparative Examples 1-2 did not show any increasing effect (results are not shown). At other growth stages, there were no significant differences between the conventional fertilization treatment in Treatment 2 and the treatments with 20% nitrogen reduction combined with the microalgae bio-fertilizers of Examples 1, 3-5, and Examples 6-7.

[0145] Test Example 2: Greenhouse Gas Emission Monitoring In-situ monitoring of soil greenhouse gas emission fluxes was conducted using a static chamber-gas chromatography method. Soil N2O emission fluxes and cumulative soil N2O emissions were calculated using methods recommended in the group standard T / LCAA006-2021, "Technical Specification for Static Chamber Method for Monitoring Methane and Nitrous Oxide Emissions from Farmland".

[0146] 2.1 Soil N2O emission flux 1) N2O emission flux during the wheat season exhibits significant seasonal variations. Throughout the growing season, soil N2O emission flux varies due to the application of exogenous carbon. Treatments with 20% nitrogen reduction plus microalgae bio-fertilizers (Examples 1, 3-5) and Examples 6-7, compared to conventional fertilization (Treatment 2), reduced N2O emission flux by approximately 46.90%. The microalgae bio-fertilizer treatments in Comparative Examples 1-2 did not show any emission reduction effect.

[0147] 2) N2O emission flux during the maize season exhibits significant seasonal variations. Throughout the growing season, soil N2O emission flux varies due to the introduction of exogenous carbon. Treatments with a 20% nitrogen reduction plus microalgae bio-fertilizer (Examples 1, 3-5) and treatments in Examples 6-7, compared to conventional fertilization (treatment 2), reduced N2O emission flux by approximately 38.20%. The microalgae bio-fertilizer treatments in Comparative Examples 1-2 did not show any emission reduction effect.

[0148] 2.2 Cumulative N2O Emissions from Soil Table 2 shows the cumulative N2O emissions from the soil during the wheat and corn seasons. As can be seen from Table 2, compared with conventional fertilization (treatment 2), the cumulative N2O emissions from the treatments with 20% nitrogen reduction plus microalgae bio-fertilizers from Examples 1, 3-5, and Examples 6-7 were significantly reduced. Compared with 20% nitrogen reduction (treatment 3), the treatments with 20% nitrogen reduction plus microalgae bio-fertilizers from Examples 1, 3-5, and Examples 6-7 further reduced the cumulative N2O emissions from the soil. However, compared with 20% nitrogen reduction (treatment 3), the treatments with 20% nitrogen reduction plus microalgae bio-fertilizers from Comparative Examples 1-2 did not show any emission reduction effect.

[0149] Table 2 Cumulative N2O Emissions from Soil Note: Different lowercase letters after the data in the same column indicate significant differences at the P<0.05 level, while the same letter indicates no significant difference.

[0150] Test Example 3: Monitoring the Wheat-Maize Yield Effect To clarify the impact of fertilizer reduction and exogenous carbon input on crop growth, development, and yield, the measured indicators should include yield and yield factors, biomass accumulation and distribution, and cost-benefit evaluation of the winter wheat-summer maize rotation system. Wheat and maize yield indicators should be calculated using methods recommended in the industry standards NY / T 1301-2025 "Technical Specifications for Crop Variety Testing and Information Technology for Wheat" and NY / T 1209-2020 "Technical Specifications for Crop Variety Testing and Information Technology for Maize".

[0151] 3.1 Wheat Season By monitoring wheat yield and its components (number of effective spikes, number of grains per spike, thousand-grain weight, and biomass), the yield effects of different fertilization management measures were compared and analyzed. The results are shown in Table 3.

[0152] Table 3. Factors influencing wheat yield under different fertilization practices As shown in Table 3, compared with conventional fertilization (treatment 2), the yields of the 20% nitrogen reduction + microalgae bio-fertilizer treatments in Examples 1, 3-5, and Examples 6-7 all showed an increasing trend; the total biomass did not differ significantly. This indicates that microalgae bio-fertilizer mainly increases yield by promoting the translocation and accumulation of photosynthetic products into the grains, rather than simply increasing the total biomass, reflecting the promoting effect of active substances such as extracellular polysaccharides, amino acids, and plant hormone analogs produced by microalgae metabolism on crop reproductive growth. However, the 20% nitrogen reduction + microalgae bio-fertilizer treatments in Examples 1-2 did not show any yield-increasing effect.

[0153] 3.2 Corn Season By monitoring maize yield and its components (number of effective ears, number of kernels per ear, thousand-kernel weight, and biomass), the yield effects of different fertilization management practices were compared and analyzed. The results are shown in Table 4. Table 4. Factors influencing maize yield under different fertilization practices As shown in Table 4, compared with conventional fertilization (treatment 2), the yields of the treatments with 20% nitrogen reduction plus microalgae bio-fertilizers from Examples 1, 3-5, and Examples 6-7 all showed an increasing trend. The increase in biomass was significant, with an increase of 40.71%; this indicates that the nitrogen-fixing capacity and bioactive substance secretion capacity of soil microalgae were further enhanced during the maize season. However, the treatments with 20% nitrogen reduction plus microalgae bio-fertilizers from Examples 1-2 did not show any yield increase.

[0154] Comprehensive comparative analysis conclusions: 1) In terms of soil carbon sequestration and soil fertility improvement, the exogenous carbon input measure of reducing nitrogen by 20% and applying microalgae bio-fertilizer can increase soil organic carbon storage, showing its excellent potential for soil sequestration and quality improvement.

[0155] 2) In terms of greenhouse gas emission reduction, compared with conventional fertilization, the treatment of reducing nitrogen by 20% and applying microalgae bio-fertilizer significantly reduced the cumulative N2O emissions and emission intensity per unit yield in farmland soil.

[0156] 3) In terms of ensuring high and stable crop yields, reducing nitrogen by 20% and applying microalgae bio-fertilizer can further increase crop yields, demonstrating good potential for stable yield and increased efficiency.

[0157] Compared with conventional fertilization, the application of microalgae bio-fertilizer with a 20% reduction in nitrogen content in this invention helps maintain stable crop yield and increase while synergistically increasing carbon sequestration and reducing emissions in the long term by increasing soil organic carbon content. Furthermore, the results show that the microalgae bio-fertilizer prepared in this invention, by controlling the volume ratio of *Trichophyton spp.* liquid to *Chlorella sorogenesis* liquid at (1~2):(1~2) and using a wheat-corn rotation fertilization method, produces a synergistic effect of carbon sequestration, emission reduction, and yield increase. When this volume ratio is too low (e.g., 1:5 in Comparative Example 1) or too high (e.g., 5:1 in Comparative Example 2), the increase in soil organic carbon content and the reduction in N2O emission flux are significantly lower than in the examples. This indicates that only when the volume ratio of *Trichophyton spp.* liquid to *Chlorella sorogenesis* liquid is within the range of (1~2):(1~2) can the two produce a synergistic effect of carbon sequestration, emission reduction, and yield increase. If the range is exceeded, the nitrogen fixation and photosynthetic carbon fixation functions will be unbalanced, and neither of them can achieve the effects of carbon fixation, emission reduction and increased production, thus failing to achieve the technical effects of the present invention.

[0158] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A fertilization method for soil carbon sequestration and emission reduction in wheat-corn rotation, characterized in that, The fertilization method includes, in a wheat-corn rotation system, after harvesting corn and wheat, crushing and returning corn and wheat straw to the field, spraying microalgae bio-fertilizer that promotes soil carbon sequestration and emission reduction on the soil surface, and applying soil conditioner and compound fertilizer after sowing wheat and corn. The method for preparing the microalgae bio-fertilizer includes the following steps: S1. Algal strain amplification culture: FACHB-319 and Chlorella sologenophylla 1230 were inoculated into the culture medium and amplified stepwise under suitable temperature and light conditions. S2, Mixing and Encapsulation: Mix the two amplified algal solutions obtained in step S1 at a volume ratio of (1~2):(1~2), add encapsulation agent, and allow algal cells to self-assemble into encapsulated spores. Let it stand in a dark environment. S3. Mesoporous silica encapsulation treatment: The encapsulated spore suspension obtained in step S2 is mixed with the mesoporous silica carrier solution and reacted under stirring to form a mesoporous shell layer on the surface of the encapsulated spores, thus obtaining a microalgae silica complex. S4. Finished product preparation: Collect the microalgae silica complex obtained in step S3, wash, resuspend, and adjust the microalgae cell concentration, and then dispense it into containers to prepare the microalgae bio-fertilizer for soil carbon sequestration and emission reduction in wheat-corn rotation. In step S3, the volume ratio of the encapsulated spore suspension to the mesoporous silica carrier solution is 1:(2~4).

2. The fertilization method according to claim 1, characterized in that, In step S1, the culture temperature is 20~30℃ and the light intensity is 2000~4000 lux.

3. The fertilization method according to claim 1, characterized in that, In step S1, when the number of upper-level microalgal cells is ≥10 7 When the number of cells / mL reaches a certain level, proceed to the next stage of culture.

4. The fertilization method according to claim 1, characterized in that, In step S2, the encapsulation agent is a controlled-release nanoparticle encapsulation agent of tea polyphenols, the concentration of the encapsulation agent is 0.5~1.5 mg / L, and the standing time is 12~36 hours.

5. The fertilization method according to claim 4, characterized in that, In step S2, the tea polyphenol controlled-release nanoparticle encapsulation agent is prepared by emulsion solvent evaporation method, which includes dissolving polylactic acid in dichloromethane, adding tea polyphenols to form the inner phase, using gelatin aqueous solution as the outer phase, and then encapsulating the product by stirring, filtering, washing and drying.

6. The fertilization method according to claim 5, characterized in that, The mass ratio of polylactic acid to tea polyphenols is (1~5):(1~5), and the mass fraction of gelatin in the gelatin aqueous solution is 0.5%~2.0%.

7. The fertilization method according to claim 1, characterized in that, The microalgae bio-fertilizer contains ≥1.0×10⁻⁶ live microalgae cells. 8 per mL.

8. The fertilization method according to claim 1, characterized in that, The process includes pouring the microalgae bio-fertilizer into water and stirring it evenly; the volume ratio of the microalgae bio-fertilizer to water is 1:(250~750), to obtain a diluted solution of the microalgae bio-fertilizer.

9. The fertilization method according to claim 8, characterized in that, The volume ratio of the microalgae bio-fertilizer to water is 1:500, and a diluted solution of the microalgae bio-fertilizer is sprayed onto the soil surface at a rate of 10-15 L / ha.

10. The application of the fertilization method as described in any one of claims 1-9 in the wheat-maize rotation system for carbon sequestration, emission reduction and yield increase.

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