Preparation and application of a biological remediation liquid taking active polymorphic triad algae as core

Abstract

A kind of biological repair liquid with active polymorphic triad algae as core is prepared and applied.It belongs to the field of agricultural biotechnology and soil remediation.Preparation: polymorphic triad algae is cultured to logarithmic growth phase, and polymorphic triad algae liquid is obtained, and after passing mirror examination, it is expanded and propagated to obtain the biological repair liquid with active polymorphic triad algae as core.Application: used for improving the physical and chemical properties of crop soil and improving soil fertility; applied at any stage from the emergence stage to the reproductive growth stage of crop growth; the application mode is to spray the soil surface around the root system of crops.The formula of the biological repair liquid in the application is simple, the culture medium is low in cost, the expansion cycle is short, the operation is simple, and the biological repair liquid can be quickly produced on a large scale.The biological repair liquid in the application maintains the continuous propagation and metabolism ability of microalgae; continuously supplies natural nutrients for crops; repairs soil, activates soil trace elements, improves plant stress resistance, prevents diseases and insect pests, promotes crop growth, improves agricultural product quality and yield.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology and soil remediation, specifically involving the preparation and application of a bioremediation solution with active and variable trichophyton as the core. Background Technology

[0002] With the improvement of people's living standards and the continuous reduction of arable land in my country, consumers' demand for agricultural products has shifted from "eating enough" to "eating well, eating healthily, and eating safely." The requirements for quality, flavor, nutritional function, and traceability safety have increased unprecedentedly, thus giving rise to high-value, high-quality, high-efficiency, and sustainable agriculture.

[0003] In traditional agriculture, when soil fertility levels are insufficient to provide the nutrients required for plant growth, fertilization is necessary. This includes chemical fertilizers, organic fertilizers, and bio-fertilizers, with chemical fertilizers currently accounting for 90% of all fertilizers used. The negative impacts of chemical fertilizer application cannot be ignored. The raw materials for chemical fertilizers contain heavy metals such as Zn, Cu, Co, and Cr. These heavy metals are introduced into the soil along with the fertilizer, causing their accumulation. They are then absorbed by crops, affecting crop growth and the quality of agricultural products. Chemical fertilizers reduce the number and activity of soil microorganisms, decreasing their ability to transform organic matter, decompose minerals, and degrade toxic substances. Long-term use of chemical fertilizers exacerbates soil acidification, soil compaction, soil degradation, and reduced soil fertility. It also inhibits root development, severely reduces crop resistance, and leads to resource waste and increased agricultural non-point source pollution.

[0004] In addressing soil degradation, increasing soil organic matter is considered one of the most effective methods. Although soil organic matter accounts for only a small portion of the total soil volume, it plays a crucial role in improving soil physical structure, regulating soil pH, enhancing soil fertility, protecting the environment, and promoting sustainable agricultural and forestry development. Organic matter plays a multifaceted role in improving soil physical properties, with its most important and direct effect being the improvement of soil structure, promoting the formation of granular structures, thereby increasing soil looseness, aeration, and permeability. Traditional methods for increasing soil organic matter mainly include applying well-rotted organic fertilizer, returning straw to the field, and planting green manure. However, each of these methods has its own drawbacks and limitations in application. For example, the sources of well-rotted organic fertilizer are not widely available; the promotion of large-scale mechanized straw return is limited, and the cost of small-scale mechanized straw return is high; planting green manure indirectly increases farmers' input costs.

[0005] Soil salinization is increasingly threatening the land resources upon which humanity depends for survival. With arable land decreasing and freshwater resources becoming scarce, how to utilize large areas of saline-alkali land and desertified land for agricultural development has become a current challenge. my country has a vast area of ​​saline-alkali land with complex types. The accumulation of large amounts of salt in the soil leads to a series of deteriorations in soil physical properties, such as sticky structure, poor aeration, slow soil temperature rise, poor activity of aerobic microorganisms, slow moisture release, low permeability, and strong capillary action, further exacerbating surface soil salinization. Furthermore, the compaction, infertility, and high pH of saline-alkali soil reduce its drought and flood resistance, disrupt its original structural composition, decrease the content of organic matter, and weaken the activity of microorganisms in the shallow soil.

[0006] The pursuit of crop yield at the expense of soil conditioning has led to severe damage to the soil environment. Soil compaction, salinization, and acidification are common problems. Furthermore, the increasingly complex natural environment, such as late spring frosts, necessitates greater plant resilience to adapt to these changes. The market demands products that can improve soil conditions and enhance plant resistance. These changes in the soil ecosystem have also altered crop quality. People often say that fruits and vegetables don't taste as good as they used to, creating a market need for products that improve crop quality. Simultaneously, due to various reasons, soil-borne diseases such as seedling death, root rot, damping-off, and wilt occur frequently. Once soil-borne diseases occur, they can cause anything from crop wilting and reduced yields to crop death and total crop failure, resulting in significant losses.

[0007] Microalgae, through their reproduction and metabolism, can degrade residual chemical fertilizers, organic pesticides, heavy metals, and other pollutants in the soil, reducing the degree of soil pollution. Microalgae are a type of autotrophic plant widely distributed in terrestrial soils, rivers, and lakes, with high photosynthetic utilization. Their morphology, structure, and species can only be identified using biological microscopes and molecular biology techniques. Microalgae are rich in nutrients; their cell metabolism produces polysaccharides, proteins, and other nutrients, making them valuable in the food, pharmaceutical, and animal feed industries. Large-scale cultivated microalgae can promote growth, reduce fertilizer use, and improve the quality of agricultural crops. However, currently, microalgae added to biofertilizers are often in a dead state, only able to utilize nutrients within their own tissues. They cannot reproduce, continuously metabolize, or colonize the rhizosphere, significantly limiting their function. Their fertilizing and remediation effects are short-lived and unstable, greatly restricting their value as fertilizers.

[0008] Microalgae, due to their high photosynthetic efficiency, wide distribution, and abundant metabolites, have great potential for application in agriculture. Nitrogen-fixing cyanobacteria, in particular, can fix atmospheric molecular nitrogen into bound nitrogen, synthesizing nutrients such as proteins. They also possess functions such as soil remediation, nutrient activation, growth promotion, and enhanced stress resistance. Existing research and applications show that nitrogen-fixing cyanobacteria can reduce fertilizer application, increase crop yields, and improve soil properties. They have demonstrated certain yield-increasing effects on crops such as rice and wheat. Some species can also inhibit soil-borne diseases such as root-knot nematodes and secrete bioactive substances such as auxins, gibberellins, and cytokinins, promoting seed germination and crop growth.

[0009] However, existing nitrogen-fixing cyanobacteria-based biofertilizers and soil remediation agents still have many insurmountable technical shortcomings:

[0010] The algal strains were not targeted for selection, and most of them were mixed algae or general algal strains, with mixed functions, weak targeting, and unstable application effects.

[0011] Most of the products are dry and inactivated algae powder. The algae lose their ability to reproduce and metabolize, and cannot colonize in the soil, continuously fix nitrogen, or secrete active substances, thus greatly reducing the repair and growth promotion effects.

[0012] Traditional cultivation processes are complex, culture medium components are cumbersome, and production costs are high, making it difficult to scale up production and promote in the field.

[0013] The algae strain has poor soil adaptability and is difficult to survive in challenging soils such as saline-alkali, acidified, compacted, and heavy metal polluted soils, thus failing to effectively improve the physical and chemical properties of the soil.

[0014] The product has a single function and it is difficult to achieve the comprehensive goals of soil remediation, fertility improvement, disease control, and stress resistance and yield increase at the same time;

[0015] It has poor compatibility with chemical fertilizers and cannot fundamentally alleviate the soil degradation problem caused by long-term application of chemical fertilizers.

[0016] In summary, existing technologies cannot meet the current agricultural demand for green, efficient, low-cost, and multifunctional soil remediation and crop growth-promoting products. Therefore, developing a nitrogen-fixing cyanobacteria preparation with high activity, strong colonization ability, wide soil adaptability, continuous function, and combined soil remediation, quality improvement, and yield enhancement functions has significant practical significance and application value. Summary of the Invention

[0017] The purpose of this invention is to solve the problems existing in the prior art and to provide a preparation and application of a bioremediation solution with active and variable trifoliate algae as the core.

[0018] A bioremediation solution based on the active and variable Trichophyton spp. is prepared by the following steps:

[0019] I. Preparation of algal solution:

[0020] Inoculate Trichormus variabilis into the culture medium and culture it until the logarithmic growth phase to obtain Trichormus variabilis algal solution, and then conduct microscopic examination to obtain the Trichormus variabilis algal solution after passing the microscopic examination;

[0021] II. Subculture and propagation:

[0022] Mix the above-mentioned Trichormus variabilis algal solution after passing the microscopic examination with the culture medium, conduct subculture and propagation, and obtain a bioremediation solution with active Trichormus variabilis as the core, thus completing the preparation;

[0023] Among them, the Trichormus variabilis described in step I is microalgae ZR2602, which has been deposited in the China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 2026567, the deposit time is March 30, 2026, and the deposit address is Wuhan University, China. It is Trichormus variabilis ZR2602.

[0024] Furthermore, the inoculation amount of the Trichormus variabilis described in step I is 10%.

[0025] Furthermore, the qualified standard for the microscopic examination in step I is that the density of the Trichormus variabilis algal solution is OD 680 = 0.610 - 0.900, and the activity is manifested as full cell shape, complete structure, active movement, and full and bright pigment color inside the cells.

[0026] Furthermore, the volume ratio of the Trichormus variabilis algal solution after passing the microscopic examination to the culture medium in step II is (3 - 5)∶1.

[0027] Furthermore, the culture media described in step I and step II are the same: both are adding 1.5 g of sodium nitrate, 40 mg of potassium dihydrogen phosphate, 75 mg of magnesium sulfate heptahydrate, 6 mg of citric acid, 6 mg of ammonium ferric citrate, 1.1 mg of disodium ethylenediaminetetraacetate, 27.2 mg of calcium chloride, 20 mg of sodium carbonate, 2.86 mg of boric acid, 1.86 mg of manganese chloride, 0.22 mg of zinc sulfate, 0.0八 mg of copper sulfate, and 0.05 mg of cobalt nitrate into each liter of sterilized water, and the pH of the culture medium is 7.0 ± 0.1; the culture medium is sterilized at 121 °C for 15 min in a high-temperature and high-pressure sterilizer.

[0028] Furthermore, the conditions for the culture in step I and the subculture and propagation in step II are the same: under the conditions of a temperature of 26 - 30 °C, a daily light illumination time of 12 - 16 h, and a light intensity of 4000 - 5500 lux, seal and culture for 4 - 7 d, and add fresh culture medium every 2 - 3 d during the culture period.

[0029] The above-mentioned bioremediation solution, with active and variable Trichophyton spp. as its core, is used to improve the physical and chemical properties of crop soil and enhance soil fertility.

[0030] Furthermore, the application of the bioremediation solution centered on the active and variable Trichophyton spp. is specifically carried out as follows: the bioremediation solution centered on the active and variable Trichophyton spp. is applied at any stage of crop growth, from the seedling stage to the reproductive growth stage; the application method is spraying onto the soil surface around the crop roots; the application rate is per 225cm³. 2 Apply 25-50 mL to the soil surface area.

[0031] The *Trichophyllaria variegata* used in this invention is a microalga ZR2602, belonging to the nitrogen-fixing cyanobacteria family. It possesses highly efficient nitrogen fixation and decomposition capabilities, fixing atmospheric molecular nitrogen into bound nitrogen and further synthesizing proteins. This process repairs the soil, activates soil micronutrients, enhances plant resistance, prevents pests and diseases, and improves the quality and yield of agricultural products. The microalgae envelops crop roots, forming a symbiotic relationship, fixing nitrogen from the air and supplying it to the roots. *Trichophyllaria variegata* can not only survive and reproduce on the surface of crop roots but also enter the plant interior, living in the roots, stems, leaves, and fruits, providing crops with rich and balanced natural nutrients. *Trichophyllaria variegata* infuses the soil with powerful vitality; its microalgal cells multiply rapidly, absorbing carbon dioxide and releasing large amounts of oxygen, creating a favorable environment for aerobic microorganisms in the soil, activating various native soil microbial communities, and improving the soil's microecological environment, making the soil vibrant and full of life.

[0032] In this invention, the variable trifoliate algae, through its photoautotrophic properties, reproduces and develops, and undergoes autotrophic metabolism, adsorbing, degrading, transforming, and solidifying pollutants such as chemical residues and heavy metal residues in the soil. It can decompose organic matter that cannot be broken down by plants, animals, or humans in extreme environments into inorganic substances, water, and nitrogen dioxide. The variable trifoliate algae can absorb and utilize these substances, thereby altering the properties and structure of the soil, regulating soil pH, and increasing soil organic matter and fertility. Furthermore, the variable trifoliate algae can utilize insoluble phosphorus and potassium, and activate insoluble calcium, magnesium, or sulfur elements, transforming them into readily absorbable forms for plants. The biostimulants produced during its life activities provide nutrients to stimulate crop growth, control or inhibit the activity of plant pathogens, enhance crop resistance, change the original form of heavy metal elements in the soil, and block heavy metals from entering the plant body through the root system, thus producing safe and healthy food for humans.

[0033] The bioremediation solution prepared in this invention, with active and variable Trichoderma as its core, contains no chemical components and will not cause the enrichment of heavy metal elements in the soil. It effectively reduces the absorption of heavy metal elements by crops, thereby improving crop growth and the quality of agricultural products. It will not reduce the number and activity of soil microorganisms, nor will it affect the transformation of organic matter, decomposition of minerals, and degradation of toxic substances by soil microorganisms. It will not cause soil acidification, compaction, or other phenomena.

[0034] The bioremediation solution prepared in this invention, with active and variable *Trichophyton spp.* as its core, serves as a nutrient-rich bio-fertilizer with excellent compatibility and symbiotic properties. It can repair soil, replenish natural nitrogen, activate soil trace elements, improve crop resistance, prevent pests and diseases, and enhance the quality and yield of agricultural products. Applying *Trichophyton spp.* to paddy fields serves two purposes: firstly, it provides a source of nitrogen fertilizer, reducing the amount of chemical nitrogen fertilizer used; secondly, the decomposition of the dead algae increases soil organic matter, comprehensively improving soil quality; and thirdly, the small size of *Trichophyton spp.* allows it to be incorporated into the soil through tilling without additional measures or costs. Combining the application of *Trichophyton spp.* with field management practices not only helps alleviate soil degradation and improve fertilizer utilization but also reduces farmers' input costs to a certain extent.

[0035] The bioremediation solution prepared in this invention, with active and variable *Trichoderma* as its core, can fix nitrogen, produce auxins and cytokinins, contains various trace elements, is highly valuable, and does not produce drug residues, thus enabling sustainable agricultural development. It has unparalleled advantages in promoting crop yield, improving crop resilience, and being pollution-free. Compared to conventional agricultural fertilizers, it not only significantly promotes crop root development and enhances crop photosynthesis, but also promotes early fruit ripening and greatly improves crop quality, especially for cash crops such as vegetables, melons, and flowers. By improving soil properties and fertility, reducing fertilizer fixation and loss, chelating chemical elements, stimulating crop growth, and improving fertilizer utilization by crops, it enhances the overall effectiveness of fertilizers.

[0036] The microalga ZR2602 in this invention is a single superior algae species that has been selectively isolated and purified. It belongs to the category of highly efficient nitrogen-fixing cyanobacteria, with well-defined functions and stable and reliable effects, solving the problems of mixed algae species and uncontrollable effects. It has a strong nitrogen-fixing function, which can fix molecular nitrogen in the atmosphere into bound nitrogen and further synthesize proteins, thereby achieving soil repair, activation of soil trace elements, improvement of plant stress resistance, prevention of pests and diseases, promotion of crop growth, and improvement of agricultural product quality and yield.

[0037] The bioremediation solution prepared in this invention, with active and variable tri-algae as its core, maintains the continuous reproduction and metabolic capacity of microalgae, completely changing the situation where dead algae can only provide limited nutrition. The variable tri-algae can coat crop roots, forming a symbiotic relationship with them. It can not only survive and reproduce on the root surface, but also enter the roots, stems, leaves, and fruits to colonize, continuously supplying crops with natural nutrients. It can grow normally in soils with obstacles such as saline-alkali, acidified, compacted, and heavy metal polluted soils. Through large-scale reproduction, it fixes carbon and releases oxygen, improves soil aeration, activates native microorganisms, and rebuilds the soil micro-ecology. It can simultaneously complete multiple functions such as soil remediation, nutrient activation, disease suppression, stress resistance and yield increase, achieving integrated comprehensive efficiency.

[0038] The bioremediation solution of this invention has a simple formula, low cost, short expansion cycle, and simple operation, and can be rapidly mass-produced.

[0039] This invention is applicable to the preparation and application of bioremediation solutions based on the variable trifoliate algae. Detailed Implementation

[0040] Specific Implementation Method 1: This implementation method describes the preparation of a bioremediation solution based on highly active and variable algae, which is carried out according to the following steps:

[0041] I. Preparation of algal solution:

[0042] The variable trifoliol was inoculated into the culture medium and cultured to the logarithmic growth phase to obtain the variable trifoliol algal solution. Then, it was examined under a microscope to obtain the variable trifoliol algal solution that passed the microscopic examination.

[0043] II. Propagation and Expansion:

[0044] Take the above-mentioned qualified microscopic examination algal solution of Trichophyton spp. and mix it with the culture medium for expansion and propagation to obtain a bioremediation solution with active Trichophyton spp. as the core, thus completing the preparation.

[0045] The variable trichormus mentioned in step one is the microalga ZR2602, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026567, deposited on March 30, 2026, at Wuhan University, Wuhan, China.

[0046] In step two of this implementation method, the large-scale culture is carried out by shaking the culture vessel at a speed of 140-160 r / min; the small-scale culture bottle culture is carried out by manually shaking the bottle 2-3 times a day to ensure sufficient agitation of the algal solution.

[0047] The bioremediation solution prepared in this embodiment, with active and variable Trichoderma as its core, should be stored in the following environment: at room temperature, in a sealed, cool, and dry place, away from light.

[0048] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the inoculum size of the variable *Trichophyton spp.* in step one is 10%. Everything else is the same as in Specific Implementation Method One.

[0049] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One in that, in step one, the microscopic examination qualification standard is that the density of the polymorphic tri-algae solution is OD. 680 =0.610~0.900, the activity is characterized by plump cell shape, intact structure, active motility, and rich and bright pigment color within the cells. Other aspects are the same as in Specific Implementation Method 1.

[0050] Specific Implementation Method Four: This implementation method differs from Specific Implementation Method One in that the volume ratio of the microscopically examined, qualified *Algae Tricoloris* solution to the culture medium in step two is (3~5):1. Everything else is the same as in Specific Implementation Method One.

[0051] Specific Implementation Method Five: This implementation method differs from Specific Implementation Method One in that the culture medium used in steps one and two is the same: 1.5g sodium nitrate, 40mg potassium dihydrogen phosphate, 75mg magnesium sulfate heptahydrate, 6mg citric acid, 6mg ferric ammonium citrate, 1.1mg disodium EDTA, 27.2mg calcium chloride, 20mg sodium carbonate, 2.86mg boric acid, 1.86mg manganese chloride, 0.22mg zinc sulfate, 0.08mg copper sulfate, and 0.05mg cobalt nitrate are added per liter of sterilized water. The pH of the culture medium is 7.0±0.1. The culture medium is sterilized at 121°C for 15 minutes in an autoclave. Everything else is the same as in Specific Implementation Method One.

[0052] Specific Implementation Method Six: This implementation method differs from Specific Implementation Method One in that the cultivation conditions described in Step One and the propagation conditions described in Step Two are the same: a temperature of 26-30℃, a daily light duration of 12-16 hours, and a light intensity of 4000-5500 lux, with sealed cultivation for 4-7 days, and fresh culture medium added every 2-3 days during the cultivation period. Everything else is the same as in Specific Implementation Method One.

[0053] Specific implementation method seven: This implementation method uses bioremediation liquid with active and variable trifoliate algae as the core. It is applied to improve the physical and chemical properties of crop soil and enhance soil fertility.

[0054] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Method Seven in that it utilizes a bioremediation solution centered on the active and variable Trichophyton spp. The specific operation is as follows: The bioremediation solution centered on the active and variable Trichophyton spp. is applied at any stage of crop growth, from seedling emergence to reproductive growth; the application method is spraying onto the soil surface around the crop roots; the application rate is per 225cm². 2 Apply 25-50 mL to the soil surface area. Everything else is the same as in Specific Implementation Method Seven.

[0055] The beneficial effects of the present invention are verified through the following embodiments:

[0056] The following description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0057] Example:

[0058] A bioremediation solution based on the active and variable Trichophyton spp. is prepared by the following steps:

[0059] I. Preparation of algal solution:

[0060] The variable trifoliol was inoculated into the culture medium and cultured to the logarithmic growth phase to obtain the variable trifoliol algal solution. Then, it was examined under a microscope to obtain the variable trifoliol algal solution that passed the microscopic examination.

[0061] II. Propagation and Expansion:

[0062] The above-mentioned qualified microscopic examination algal solution of Algae Trichophyton spp. is mixed with culture medium and propagated to obtain a bioremediation solution with active Algae Trichophyton spp. as the core, thus completing the preparation.

[0063] Several microalgae strains were extracted from high-yield rice paddies in Qiqihar, Heilongjiang Province. After separation, purification, and propagation, repeated experiments were conducted to finally select a strain with strong nitrogen-fixing ability, capable of degrading heavy metals, pesticides, and other harmful substances, fixing carbon, providing oxygen, and secreting various bioactive substances such as carotenoids, proteins, fatty acids, plant hormones, extracellular polysaccharides, vitamins, and antibiotics. This strain can improve plant stress resistance and crop quality, repair soil compaction, increase organic matter, repair salinization, and has strong photosynthetic capacity, is rich in nutrients, and easily survives in soil. This strain is *Trichophyton variegata* (i.e., microalgae ZR2602). After propagation with culture medium, a bioremediation solution with active *Trichophyton variegata* as its core was obtained.

[0064] In step 1 of this embodiment, the Trichormus variabilis is the microalgae ZR2602, which has been deposited in the China Center for Type Culture Collection with the deposit number: CCTCC NO: M 2026567, the deposit date is March 30, 2026, and the deposit address is Wuhan University, China. It is Trichormus variabilis ZR2602.

[0065] In step 1 of this embodiment, the Trichormus variabilis is the microalgae ZR2602. Under microscopic examination, the algal filaments are curved, constricted at the transverse walls, and the apical cells at the ends are blunt-rounded; the cells are barrel-shaped, 2.1 - 3.7 μm wide and 2.5 - 6.0 μm long; the heterocysts are oblong, 3.0 - 4.0 μm wide and 4.0 - 6.0 μm long.

[0066] In step 1 of this embodiment, the inoculation amount of the Trichormus variabilis is 10%.

[0067] In step 1 of this embodiment, the qualified microscopic examination: the standard is that the density of the Trichormus variabilis algal solution is OD 680 = 0.610 - 0.900, and the activity is manifested as plump cell shape, complete structure, active movement, and bright and vivid pigment color inside the cells.

[0068] In step 2 of this embodiment, the volume ratio of the Trichormus variabilis algal solution after qualified microscopic examination to the culture medium is (3 - 5)∶1.

[0069] In steps 1 and 2 of this embodiment, the culture media are the same: 1.5 g of sodium nitrate, 40 mg of potassium dihydrogen phosphate, 75 mg of magnesium sulfate heptahydrate, 6 mg of citric acid, 6 mg of ammonium ferric citrate, 1.1 mg of disodium ethylenediaminetetraacetate, 27.2 mg of calcium chloride, 20 mg of sodium carbonate, 2.86 mg of boric acid, 1.86 mg of manganese chloride, 0.22 mg of zinc sulfate, 0.08 mg of copper sulfate, and 0.05 mg of cobalt nitrate are added to each liter of sterilized water. The pH of the culture medium is 7.0 ± 0.1; the culture medium is sterilized at 121 °C for 15 min in a high-temperature high-pressure sterilizer.

[0070] In this embodiment, the microalgae ZR2602 has a high demand for nutrients such as carbon, nitrogen, and phosphorus. Therefore, through scientific formula design of the culture medium, it ensures the sufficient supply and reasonable ratio of nutrients required for the growth of algae, can significantly promote the growth and metabolism of algae, and improve the quality and yield. Through fine nutritional regulation and metabolic optimization, the precise control of the algal growth environment is achieved, the metabolic pathway of algae is optimized, and the photosynthesis efficiency and material synthesis ability are improved. At the same time, by regulating different light-dark ratios and temperature ratios, the growth performance and stress resistance of the microalgae ZR2602 are further improved.

[0071] The cultivation conditions described in step one and the propagation conditions described in step two of this embodiment are the same: the temperature is 28℃, the daily light duration is 14h, and the light intensity is 5000 lux, and the culture is sealed and cultured for 7 days, with fresh culture medium added every 2 days during the cultivation period.

[0072] In step two of this embodiment, the propagation and small-scale culture in flasks are carried out by manual shaking three times a day to ensure sufficient agitation of the algal solution.

[0073] The application of bioremediation solutions based on highly active and variable algae is used to improve the physical and chemical properties of crop soils and enhance soil fertility.

[0074] This embodiment utilizes a bioremediation solution centered on the active and variable Trichophyton spp. The specific operation is as follows: The bioremediation solution, centered on the active and variable Trichophyton spp., is applied at any stage of crop growth, from seedling emergence to reproductive growth; the application method is spraying onto the soil surface around the crop roots; the application rate is per 225cm². 2 Apply 25-50 mL to the soil surface area.

[0075] Application examples:

[0076] 1. The experiment lasted 30 days, using 5L transparent plastic buckets for cultivation, with a soil depth of 20cm and a soil surface area of ​​225cm². 2 Three treatment groups were set up: Group 1 was a blank control group, and Groups 2 were sprayed with OD at the three-leaf stage of radish roots and on the soil surface around the roots. 680 25 mL of a bioremediation solution with a concentration of 0.610 and centered on the active and variable Algae Trichophyton, was sprayed onto the soil surface around the roots of three groups at the three-leaf stage of radish. 680 50 mL of a bioremediation solution with a concentration of 0.610 and centered on the active and variable Trichophyton spp.

[0077] Radish growth was observed, and soil organic matter, available nitrogen, available phosphorus, available potassium, and pH were measured. The results are shown in Table 1. Based on the measured data and radish growth, application of a bioremediation solution centered on the active and variable *Trichophyton spp.* increased soil organic matter content, with the amount increasing with the application rate. Soil available nitrogen content significantly increased, with groups 2 and 3 showing higher levels than group 1. Available phosphorus and available potassium in group 2 increased slightly compared to the control group, while in group 3, although they decreased slightly, the overall soil nutrient supply capacity improved. Simultaneously, all three treatments slightly lowered the soil pH, bringing it closer to the neutral range. In summary, the bioremediation solution centered on the active and variable *Trichophyton spp.* can effectively improve soil physicochemical properties and enhance soil fertility, exhibiting a positive regulatory effect on radish growth.

[0078] Table 1

[0079]

[0080] 2. The experiment lasted for 30 days. Bok choy was grown in 5L transparent plastic pots, with a soil depth of 20cm and a soil surface area of ​​225cm². 2 A blank control group and a treatment group were set up. The treatment group was treated with OD245 at the seedling stage and the granulation stage of pak choi, respectively. 680 A bioremediation solution with a concentration of 0.610, centered on the active and variable *Trichophyton spp.*, was used to observe plant growth, measure soil pH, electrical conductivity, nitrogen, phosphorus, and potassium content, and plot yield. The results are shown in Table 2. The electrical conductivity of the treatment group was significantly lower than that of the control group. Soil electrical conductivity refers to the soil's ability to conduct electric current. It not only provides an important basis for the improvement of saline-alkali soils but also serves as a comprehensive indicator of fertility for non-saline-alkali soils. Higher electrical conductivity indicates higher water-soluble salt content in the soil, which harms crop growth and leads to land degradation. Therefore, a bioremediation solution centered on the active and variable *Trichophyton spp.* can repair soil salinization and improve fertility and yield.

[0081] Table 2

[0082]

[0083] 3. Disease control and crop growth promotion:

[0084] Nematode growth depends on a specific rhizosphere environment. In this embodiment, the bioremediation solution with active and variable Trichoderma spp. as its core can inhibit nematodes by altering the soil environment. This is because Trichoderma spp. (i.e., microalga ZR2602) can secrete extracellular enzymes that can directly degrade the main components of the nematode body wall (such as chitin) and eggshell, destroying their physical structure and rendering them incapable of infecting. This, in turn, controls soil nematodes, reduces the nematode infection rate, and increases crop yield. In addition, the bioremediation solution with active and variable Trichoderma spp. as its core can improve the overall soil nutrient supply capacity, making plants more resistant due to sufficient nutrition, and making it more difficult for plant root tissues to be invaded and colonized by root-knot nematodes J2s.

[0085] In this embodiment, the bioremediation solution is based on the active and variable Trichoderma solani. Trichoderma solani (i.e., microalga ZR2602) can synthesize and secrete a variety of growth-promoting substances, such as auxin, gibberellin and cytokinin. These bioactive substances play an important role in plant development, plant metabolism and plant growth regulation, and can promote seed germination, seedling growth, improve fruit quality and crop yield.

Claims

1. A preparation method for a bioremediation solution based on highly active and variable algae, characterized in that, It proceeds in the following steps: I. Preparation of algal solution: The variable trifoliol was inoculated into the culture medium and cultured to the logarithmic growth phase to obtain the variable trifoliol algal solution. Then, it was examined under a microscope to obtain the variable trifoliol algal solution that passed the microscopic examination. II. Propagation and Expansion: Take the above-mentioned qualified microscopic examination algal solution of Trichophyton spp. and mix it with the culture medium for expansion and propagation to obtain a bioremediation solution with active Trichophyton spp. as the core, thus completing the preparation. The variable trichormus mentioned in step one is the microalga ZR2602, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026567, deposited on March 30, 2026, at Wuhan University, Wuhan, China.

2. The preparation method of a bioremediation solution based on active and variable *Trichophyton spp.* as described in claim 1, characterized in that... The inoculum size of *Trichophyton spp.* in step one is 10%.

3. The preparation method of a bioremediation solution based on active and variable *Trichophyton spp.* as described in claim 1, characterized in that... The qualified microscopic examination in Step 1: The standard is that the density of the Triparma varians algal solution is OD 680 = 0.610 - 0.900, and the activity is manifested as plump cell shape, complete structure, active movement, and bright and vivid pigment color inside the cells.

4. The preparation method of a bioremediation solution based on active and variable *Trichophyton spp.* as described in claim 1, characterized in that... In step two, the volume ratio of the microscopically examined and qualified polymorphic algae solution to the culture medium is (3~5):

1.

5. The preparation method of a bioremediation solution based on active and variable algae as the core according to claim 1, characterized in that, The culture medium described in steps one and two is the same: 1.5g sodium nitrate, 40mg potassium dihydrogen phosphate, 75mg magnesium sulfate heptahydrate, 6mg citric acid, 6mg ferric ammonium citrate, 1.1mg disodium EDTA, 27.2mg calcium chloride, 20mg sodium carbonate, 2.86mg boric acid, 1.86mg manganese chloride, 0.22mg zinc sulfate, 0.08mg copper sulfate, and 0.05mg cobalt nitrate are added per liter of sterile water. The pH of the culture medium is 7.0±0.

1. The culture medium is sterilized in a high-temperature autoclave at 121℃ for 15min.

6. The preparation method of a bioremediation solution based on the active and variable *Trichophyton spp.* as described in claim 1, characterized in that... The conditions for cultivation in step one and propagation in step two are the same: the temperature is 26~30℃, the daily light duration is 12~16h, and the light intensity is 4000~5500 lux. The culture is sealed and cultured for 4~7 days, and fresh culture medium is added every 2~3 days during the cultivation period.

7. The application of the bioremediation solution prepared as described in claim 1, with active and variable *Trichophyton spp.* as its core, characterized in that... It is used to improve the physical and chemical properties of crop soil and enhance soil fertility.

8. The application of a bioremediation solution based on the active and variable *Trichophyton spp.* as described in claim 7, characterized in that... The application of the bioremediation solution centered on the active and variable Trichophyton spp. is as follows: The bioremediation solution, centered on the active and variable Trichophyton spp., is applied at any stage of crop growth, from seedling emergence to reproductive growth; the application method is spraying onto the soil surface around the crop roots; the application rate is per 225cm². 2 Apply 25-50 mL to the soil surface area.

Images