Microalgae biological soil remediation agent and preparation method thereof

Abstract

The invention belongs to the technical field of soil remediation, and discloses a microalgae biological soil remediation agent and application thereof. A sustainable benign underground micro-ecosystem is constructed, the prepared microalgae biological soil remediation agent takes a porous active carrier with positive charges as a matrix, the porous active carrier is a porous carrier obtained by compounding silicon dioxide on montmorillonite, and the porous carrier is modified to obtain an aminated porous carrier with negative charge NH2-groups between layers. And finally, chitosan cations loaded on the carrier become a porous active carrier with positive charges, and the carrier is loaded with a large amount of chlorella pyrenoidosa, so that the environmental sensitivity is reduced, the carrier can survive and breed in a harsh soil environment, the soil fertility can be remarkably improved, and the yield and quality of crops are improved. Meanwhile, the effect of reducing soil heavy metal pollution is also achieved; the microbial community diversity of the soil can be increased, the soil aggregate structure is improved, and the physical and chemical environment of the soil is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of soil remediation, and in particular to a preparation method and application of a microalgae biological soil remediation agent. Background Art On average, soil contains less than 1% organic matter, and it takes 100 years for a natural increase of 1%. Excessive exploitation leads to vegetation destruction and soil erosion, and ecological restoration could take over a thousand years. A lack of organic matter in the surface soil leads to an imbalance in nutrient distribution. Capillary pores in the soil are scarce, resulting in poor aeration and water permeability, and low microbial activity. This results in excessive or insufficient soil acidity, leading to poor water and fertilizer retention and air permeability, making it difficult to meet the actual growth needs of crops. Furthermore, industrial pollution damages the soil, exacerbating the depletion of soil organic matter.

[0002] Without organic matter and microorganisms, the soil's aggregate structure is destroyed. Even if more nitrogen, phosphorus, and potassium are applied, it will be of no use. Although there may be a lot of nitrogen, phosphorus, and potassium in the soil, they cannot be effectively absorbed and utilized by crops. This is because most of the nutrients added to the soil are lost, with an actual utilization rate of only 30% on average. Most of them flow into rivers and lakes with farmland drainage, which not only pollutes the environment but also endangers the safety and health of humans and life. In addition, the soil still lacks nutrients, fertility, and activity, and may also have a series of problems such as soil acidification, compaction, trace element deficiency, and excessive heavy metals. The root secretions and decomposition products of crops (such as parahydroxybenzoic acid PA) can inhibit the growth and activity of the roots of continuously cropped plants, and have the strongest self-toxic effect, resulting in a reduction in the content of beneficial microorganisms in the soil, causing an imbalance of microorganisms in the soil, and thus leading to continuous cropping problems in the growth process of crops. In order to effectively ensure the potential fertility of the soil, more and more regions and countries have adopted the practice of adding a large number of beneficial microorganisms to the soil, thereby better ensuring the activity of microorganisms in the soil and achieving comprehensive improvement of soil nutritional conditions. However, there are few research reports on the use of algae active cell biofertilizer.

[0003] Chinese patent publication number CN111215442B discloses a soil remediation method, providing a soil remediation method that covers the soil to be remediated with water, then airs the covered soil, applies a signaling agent and a restoring agent, and applies the restoring agent at least once after crops planted in the soil to be remediated emerge. The restoring agent comprises deep-sea fish protein, soy lecithin, alfalfa meal, aloe vera, and organic seaweed, while the signaling agent comprises shellfish, grains, and spherical algae. The soil to be remediated is first flooded to suppress dormant microbial eggs, causing them to die. The signaling agent is then used to rapidly awaken and activate the subsurface microorganisms, enabling them to work together. The restoring agent creates an environment conducive to microbial survival, improves aggregate structure, purifies and transports nutrients, and supplies crop growth. Crop root secretions nourish the microorganisms, forming a mutually beneficial, sustainable, and benign subsurface microbial ecosystem. While this invention mentions the formation of a sustainable, benign subsurface microbial ecosystem, it does not provide a method for maintaining this ecosystem, nor does it address the issue of the activity of spherical algae and fermented products in high-temperature, harsh outdoor soils.

[0004] Chinese patent publication number CN109401977B discloses an active microalgae nutrient remediation solution and its preparation method. It discloses separately expanding the cultivation of the green alga Chlorella pyrenoidosa and the nitrogen-fixing cyanobacteria Anabaena flos-aquae to obtain two algae solutions; these solutions are then mixed to produce the active microalgae nutrient remediation solution. The active microalgae nutrient remediation solution provided in this patent has the effects of repairing soil, replenishing natural nitrogen, activating soil trace elements, improving plant resistance, preventing pests and diseases, and increasing the quality and yield of agricultural products. However, the patent does not address the duration of activity of these microalgae during the soil remediation process, nor how they can maintain their vitality and continue to repair the soil in deep soil layers with insufficient light and energy.

[0005] Microalgae bioremediation utilizes microalgae to enrich and fix heavy metals in soil through bioaccumulation, cell surface adsorption, and biomineralization. Microalgae are autotrophic organisms with strong carbon and nitrogen fixation capabilities, making them beneficial for the ecological restoration of heavy metal-contaminated soils. The bioaccumulation process relies on the cellular metabolism of microalgae. Heavy metal ions are transported or diffused across the cell membrane by specialized carrier proteins, then bind to internal organelles or compounds, resulting in their fixation. This slow and irreversible process occurs only within living cells. However, during soil remediation, particularly in deep soil layers (below 10 cm), microalgae can become inactivated due to a lack of light and energy. Furthermore, in the surface soil, due to high temperatures, microalgae cannot remain active for long periods of time. Therefore, maximizing the utilization of microbial remediation to not only provide sufficient organic matter but also better maintain the activity of soil microorganisms, ensure the microalgae's strong environmental tolerance and rapid growth in the soil, and ultimately, establish a sustainable and benign underground microbial ecosystem remains a challenge. Summary of the Invention To address the above-mentioned issues, the present invention aims to provide a method for preparing a microalgae-based biological soil remediation agent, thereby resolving one or more technical issues existing in the prior art and at least providing a beneficial alternative or creating a condition. The present invention also aims to provide a method for preparing a biological soil remediation agent that can be used to remediate soil, increase soil microbial community diversity, improve soil aggregate structure, and enhance the soil's physical and chemical environment.

[0006] One object of the present invention is to provide a biological microalgae soil remediation agent, which comprises a porous active carrier and biological microalgae loaded in the porous active carrier, wherein the biological microalgae comprises a pyrenoid chlorella liquid, and the pyrenoid chlorella density is 6×10 6 cfu / g or more; the porous active carrier is a porous carrier obtained by compounding silica on montmorillonite, and the porous carrier is modified to obtain a negatively charged NH2 between the layers - The porous carrier is aminated with chitosan cations and finally loaded with chitosan cations to become a positively charged porous active carrier.

[0007] The porous carrier is a porous carrier obtained by compounding silicon dioxide on montmorillonite. Specifically, calcium-based montmorillonite is added to deionized water, and an excess of KCl solution (1 mol / L) is added, and the mixture is stirred for 10 to 12 hours to allow Ca 2+ K + Replacement, washed with deionized water until free of Cl -After centrifugation, ethanol is added and stirred evenly to fully disperse the potassium montmorillonite. The temperature is raised to 40°C, octadecyltrimethylammonium chloride is added, stirred thoroughly, and reacted for 1 to 2 hours. The suspension is taken out and centrifuged at a speed of 3500 rpm, washed with deionized water 2 to 3 times, and centrifuged again. The solid mixture at the bottom is taken out, dried and ground at 60°C to obtain modified potassium montmorillonite; the mass ratio of the octadecyltrimethylammonium chloride to the potassium montmorillonite is 1:2.

[0008] In K + The concentration is high and its hydrated ion radius is smaller than that of Ca 2+ Therefore, by extending the stirring time, the calcium-based montmorillonite can be replaced with potassium-based montmorillonite. This replacement not only improves its dispersibility, but also allows the K ion solution to be used as a nutrient for microalgae. Modifying potassium-based montmorillonite with octadecyltrimethylammonium chloride not only increases the interlayer spacing, but also significantly reduces the montmorillonite's swelling problem when exposed to water, making it more stable and with a larger interlayer spacing.

[0009] TEOS, ethanol, and deionized water are mixed in a molar ratio of 1:4:4 at 40-50°C, diluted hydrochloric acid is added dropwise to adjust the pH to 4-5, and the mixture is stirred to form a transparent sol; modified potassium montmorillonite is slowly added, stirred for 2-3 hours, heated to 60°C, allowed to stand for 12 hours, then cooled to room temperature and allowed to stand for 24 hours, centrifuged and filtered to obtain gel microspheres, which are washed with water 2-3 times, dried at 60°C, heated to 400-500°C at 2°C / min, and kept warm for 3 hours to obtain a porous carrier; the mass ratio of the transparent sol to potassium montmorillonite is 1:8.

[0010] Furthermore, the porous support is modified to obtain negatively charged NH2 - The aminated porous carrier of the present invention is specifically: At room temperature, the porous carrier is ultrasonically dispersed into toluene 2 to 3 times the weight of the porous carrier, then the temperature is raised to 80 to 90 ° C, and (3-methylaminopropyl) trimethoxysilane is slowly added under stirring, and stirring is continued for 5 to 6 hours; after the reaction is completed, it is cooled to room temperature, centrifuged, washed with a large amount of ethanol and water, and dried to obtain a negatively charged NH2 - The aminated porous carrier comprises a group; wherein the mass ratio of (3-methylaminopropyl)trimethoxysilane to modified montmorillonite is 2 to 3:5; Further preparing a positively charged porous active carrier, specifically: At a temperature of 50-60°C, chitosan is prepared into a chitosan solution with a concentration of 3-5% using a 0.05% citric acid aqueous solution, and an aminated porous carrier is added to the chitosan solution and stirred for reaction for 80-100 minutes. After the reaction is completed, glucose and sodium alginate are added and the stirring reaction is continued for 50-80 minutes. Finally, the mixture is filtered, dried, ground, and filtered through a 60-mesh filter to obtain a positively charged porous active carrier. The mass ratio of the chitosan, sodium alginate, glucose, and aminated porous carrier is 3-8:15-20:10-15:40-50. Finally, a microalgae biological soil remediation agent was prepared, specifically: Chlorella pyrenoidosa was inoculated into BG11 culture medium at an inoculation rate of 5% to 15% and an initial density of 6 × 10 6 The culture is carried out at a temperature of 20-25°C, pH=6.5-7.1, light intensity of 2000-3000 lux, and 24 hours of light per day for 3-5 days to obtain a pyrenoid Chlorella liquid, wherein the number of viable bacteria of the pyrenoid Chlorella is ≥10 9 CFU / g; A positively charged porous active carrier is added to the protein core Chlorella liquid, mixed evenly to obtain a dispersion, and after standing for 1 to 2 hours, the precipitated part is washed with deionized water and freeze-dried to obtain a microalgae biological soil remediation agent; wherein the mass ratio of the positively charged porous active carrier to the protein core Chlorella liquid is 2 to 3:10.

[0011] Chlorella pyrenoidosa is a green algae with a strong photosynthetic capacity and an extraordinary ability to absorb solar energy. It can photosynthesize in the presence of visible light. It also has a strong reproductive capacity and reproduces asexually, with a single cell releasing four new daughter cells each time it reproduces. It can grow through photoautotrophy, chemoheterotrophy, and mixotrophy. Compared to photosynthetic autotrophy, heterotrophic cultivation overcomes the problem of insufficient light, achieving higher production efficiency while reducing external pollution and ensuring better quality microalgae products. Heterotrophic cultivation allows for precise control of growth conditions and is more conducive to inducing the efficient production of specific metabolites. However, when microalgae bioremediation agents are applied to soil, especially acidic and infertile soils, the microalgae not only face challenges such as insufficient light intensity to activate photosynthetic autotrophy, but also a lack of external energy support from the surrounding environment. Furthermore, they face the challenges of high surface temperatures and harsh environmental conditions, which can quickly inactivate the applied microalgae.

[0012] Soil physical and chemical properties include soil structure and soil pH. Soil structure is essentially the accumulation and combination of soil particles in different arrangements, forming aggregates. Different arrangements often result in different soil structures. Long-term overuse of chemical fertilizers can reduce soil microbial life and lead to severe soil compaction. Soil compaction alters soil properties, impairs soil porosity, and thus affects soil fertility and tillage capacity. Soil acidification refers to an increase in hydrogen ions in the soil, a decrease in pH, and a strong or extremely strong acidity. Substances that contribute to soil acidification include acid rain from air pollution, organic acids produced by the decomposition of organic matter in the soil, and carbonic acid produced by the metabolism of soil microorganisms and plant roots. Using microalgae liquid fertilizer can improve soil aggregate structure, enhance soil physical properties, and reduce soil particle loss. Under certain conditions, it can also contribute to the formation of humus. Microalgae photosynthesis consumes large amounts of carbon sources, including carbon dioxide. Organic acids decomposed by microorganisms, such as carbonic acid, acetic acid, succinic acid, and citric acid, serve as carbon sources for the microalgae to synthesize organic matter. However, because the soil lacked organic matter and was severely clogged before it was repaired, the applied microalgae biological soil remediation agent could not survive in such a soil environment and quickly became inactive. The surface temperature was high, and the pore conditions of the shallow soil in the clogged layer were poor, so the light could not reach the intensity required for the microalgae to start autotrophy. Therefore, a certain cultivation environment needs to be provided so that the applied microalgae can overcome the actual soil conditions and be self-sufficient in harsh soil environments, achieving rapid growth and lasting activity.

[0013] Studies have shown that there is a negative charge NH2 between the layers -The protonation or deprotonation reactions at the montmorillonite edge sites and the proton exchange reactions at the base sites within the aminated porous carrier impart the ability to buffer pH, enabling it to survive and reproduce in low or high pH environments, reducing the environmental sensitivity of the Chlorella pyrenoidosa liquid it carries. Furthermore, the montmorillonite, compounded with silica, improves its light transmittance, allowing it to absorb abundant light even in the surface soil, thereby initiating a photosynthetic autotrophic mode. Deeper in the soil, because the microalgae are carried within the porous active carrier, the energy absorbed and carried by the carrier can support heterotrophic and mixotrophic growth, maintaining higher activity and fostering a sustainable and benign underground microecosystem. Furthermore, during the microalgae's growth process, the pH can be adjusted to a near-neutral state, effectively counteracting soil acidification. Furthermore, microscopic observations have shown that the microalgae liquid fertilizer improves soil aggregate structure and increases chlorophyll content in the surface soil. Soil aggregate structure determines plant rooting and establishment, as well as water and nutrient absorption, and is a primary driver and indicator of soil respiration rate and water retention capacity. Microalgae produce carbohydrates that combine with plant mucilage, mineral embryos, and organic colloids to improve soil aggregate structure. Therefore, when Chlorella protothecoides and its metabolites are applied to the soil, the algal cells can establish and grow in the soil and within plant roots. During their reproduction, they produce oxygen for root respiration, increasing soil porosity and preventing soil compaction. The quality of soil aggregate structure is often directly related to plant growth and the health and sustainability of the soil. Soil with an overly dense aggregate structure can easily lead to oxygen deficiency, especially in the substratum, creating a heterotrophic environment. This alters soil properties and pH, shifting the ecosystem towards anaerobic conditions rather than a coexistence of aerobic and anaerobic conditions, leading to decreased plant root vitality and even necrosis. The microalgae cells in microalgae-based soil remediation fertilizers are primarily single-celled or loosely bound. These cells can penetrate the soil, modulating the activity of soil microorganisms, accelerating the decomposition of soil clumps, and effectively mitigating the problem of overly dense soil aggregate structure. In the deep soil where the light is extremely weak, even if the microalgae cannot carry out photosynthetic autotrophy due to insufficient light and gradually die due to nutrient depletion, they will provide energy for other heterotrophic microorganisms and continue to contribute to the symbiotic system of the plant roots.

[0014] The present invention also aims to provide a soil remediation method based on the aforementioned biological soil remediation agent, which is specifically as follows: Mix the microalgae biological soil remediation agent and the nutrient solution evenly in a mass ratio of 8 to 10:80.

[0015] The nutrient solution includes water and a culture component accounting for 0.1-0.18% by weight; the culture component includes sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, citric acid, ammonium ferric citrate, disodium EDTA, sodium carbonate and trace elements, and the weight ratio of sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, citric acid, ammonium ferric citrate, disodium EDTA, sodium carbonate and trace elements is (110-130):(3-3.4):(5-8):(2.6-3):(0.45-0.5):(0.45-0.5):(0.05-0.09):(1.3-1.8):(0.04-0.06). Furthermore, the trace elements include H3BO3, MnCl2·4H2O, ZnSO4·7H2O, Na2MoO4·2H2O, CuSO4·H2O and Co(NO3)2·6H2O, and the weight ratio of H3BO3, MnCl2·4H2O, ZnSO4·7H2O, Na2MoO4·2H2O, CuSO4·H2O and Co(NO3)2·6H2O is (2.8~2.9):(1.8~1.9):(0.2~0.23):(0.35~0.4):(0.06~0.08):(0.03~0.06).

[0016] When the culture component accounts for 0% by weight in the nutrient solution, the method comprises the following steps: dispersing and diluting the microalgae biological soil remediation agent; drip irrigation of the obtained dispersion; When the culture component accounts for more than 0% by weight of the nutrient solution, the method includes the following steps: mixing the microalgae biological soil remediation agent with the nutrient solution, and after uniform mixing, covering the corresponding container; further, the covered container has a ventilation gap; further, the container can be a white container with a certain degree of transparency, such as a translucent white container; placing the microalgae in the corresponding container for fermentation for 2 to 3 days at a fermentation temperature of 25 to 35°C. During the fermentation process, the container is opened at least once a day for stirring, and then the container is covered and fermented. After the fermentation is completed, the corresponding container is opened to obtain a microalgae propagation solution; and the microalgae propagation solution is applied to the soil for soil remediation.

[0017] By combining the biological soil remediation agent provided by the present invention and the corresponding expansion method, the microorganisms contained in the microalgae biological soil remediation agent can be quickly and effectively expanded, thereby obtaining a large amount of applicable microbial fertilizer based on a small amount of microbial seed liquid. This facilitates the industrial application of photosynthetic microbial liquid fertilizer and reduces cost consumption. After applying the biological soil remediation agent of the present application, it is beneficial to inoculate and multiply probiotics and native soil microalgae with high activity in agricultural soil, increase the diversity of soil microbial communities, improve soil aggregate structure, and improve the soil physical and chemical environment. Compared with the prior art, the beneficial effects of this invention are: 1) The present invention addresses the problem in the prior art that microalgae have poor biological activity and cannot grow sustainably under harsh soil conditions. It constructs a sustainable and benign underground micro-ecosystem and provides a microalgae biological soil remediation agent. The biological microalgae soil remediation agent uses a positively charged porous active carrier as a matrix. The porous active carrier is obtained by compounding silicon dioxide on montmorillonite, and the porous carrier is modified to obtain a negatively charged NH2 - The porous carrier is then aminated with chitosan cations, creating a positively charged porous active carrier. This carrier supports a large amount of Chlorella pyrenoidosa, enabling it to survive and reproduce in both low and high pH environments, reducing the environmental sensitivity of the Chlorella pyrenoidosa liquid. Furthermore, the montmorillonite, compounded with silica, improves its light transmittance, allowing it to capture sufficient light even in the surface soil, thereby initiating a photosynthetic autotrophic mode. Deep in the soil, because the microalgae are loaded into the porous active carrier, the energy absorbed and carried by the carrier can support their heterotrophic and mixotrophic growth, maintaining a higher level of activity and contributing to the formation of a sustainable and benign underground micro-ecosystem. Furthermore, under the protection of the porous active carrier, the microalgae can maintain their activity even when surface temperatures rise excessively in summer, significantly improving their survival rate under high temperatures. Furthermore, the carrier can adjust the pH to a near-neutral state during the microalgae's growth process, counteracting soil acidification. This microalgae biological soil remediation agent can penetrate into the soil, overcoming the problem of persistent activity under insufficient light conditions. It can significantly regulate the activity of deep soil microorganisms, accelerate the decomposition of soil blocks, and effectively alleviate the phenomenon of overly tight soil aggregate structure.

[0018] 2) The pyrenoid Chlorella vulgaris in the microalgae biological soil remediation agent provided by this invention is highly pure and can be used directly as a nutrient fertilizer or as an algae seed for propagation. This product is easy to use, and the resulting propagation solution is highly concentrated, highly vigorous, and rapidly reproduces, making it economical and affordable. This ensures a high success rate for propagation in farmland, offering safe, stable use, convenient operation, and low cultivation costs. The pyrenoid Chlorella metabolites produce high-protein nutrients, providing crops with abundant nitrogen, phosphorus, potassium, and other nutrients that reach the rhizosphere (the soil within 1-2 mm of the root surface). Metabolites stimulate the activity of rhizosphere organisms, rapidly promoting root growth and branching, strengthening and revitalizing root hairs, and improving nutrient absorption and supply capacity. This leads to faster greening and better crop growth, resulting in higher yields and better economic benefits. Furthermore, the agent is environmentally friendly. The microalgae propagation solution maintains a pH of approximately 8.5, which regulates acidic soils. This is particularly effective in farmland with continuous cropping, increasing soil microbial diversity, promoting the growth of beneficial bacterial communities, and helping to restore the balance of the soil microbial ecosystem. When Chlorella protothecoides and its metabolites are applied to the soil, the algae cells can colonize and grow in the soil and plant roots. During the reproduction process, they will produce oxygen for the roots to breathe, increase the soil porosity and prevent the soil from becoming compacted.

[0019] 3) The microalgae biological soil remediation agent and the corresponding soil remediation method provided by the present invention can increase the diversity of soil microbial communities, improve soil aggregate structure, and improve soil physical and chemical environment. It can increase the oxygen content and organic matter in the soil, promote the formation of dominant bacterial communities of beneficial soil microorganisms, solve problems such as soil compaction, acidification, deterioration of agricultural product quality, years of crop failure, pests and diseases, thereby promoting healthy plant growth, increasing crop yields, restoring crop properties and improving crop quality. It can improve the soil to achieve effects such as root growth and seedling strengthening, nitrogen fixation and quality improvement, acid regulation and antibacterial properties. It can be used in a variety of ways, including dilution, dispersion and expansion use; in particular, the expansion method can quickly and effectively expand the microorganisms in the biological soil remediation agent, and then obtain a large amount of applicable microbial liquid fertilizer based on a small amount of initial microbial seed liquid.

[0020] 4) The biological soil remediation agent provided by this invention has the ability to degrade toxic and hazardous substances. Through the growth and proliferation of living microorganisms contained in the agent, it breaks down organic matter in the soil into small molecules that are easily absorbed by plants. This increases the supply of plant nutrients, promotes plant growth, and improves the quality of agricultural products and the agroecological environment. It features strong strain activity, high concentration, rapid propagation, and is easy to use and cost-effective. It can activate soil, enhance photosynthesis, and improve crop yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 , a comparison of soil conditions in the demonstration area fertilized with the microalgae biological soil remediation agent prepared in Example 3 and the control area fertilized with ordinary fertilizers on the 140th day; Figure 2, comparison chart of sweet potato root growth 8 days after fertilization; Figure 3 , comparison chart of sweet potato root growth 36 days after fertilization; Figure 4 , comparison chart of sweet potato harvest; Figure 5 , Growth change diagram of sweet potato after fertilizing with the microalgae biological soil remediation agent prepared in Example 3; Figure 6 , a comparison chart of rice growth conditions. DETAILED DESCRIPTION

[0022] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application. Example 1 A biological microalgae soil remediation agent comprises a porous active carrier and biological microalgae loaded in the porous active carrier, wherein the porous active carrier is a porous carrier obtained by compounding silicon dioxide on montmorillonite, specifically: 50 parts of calcium-based montmorillonite are added to 30 parts of deionized water, an excess of 1 mol / L KCl solution is added, and stirring is continued for 12 hours to allow Ca 2+ K + Replacement, washed with deionized water until free of Cl - After centrifugation, 50 parts of ethanol are added and stirred evenly to fully disperse the potassium montmorillonite, the temperature is raised to 40°C, 25 parts of octadecyltrimethylammonium chloride are added, the mixture is fully stirred, and the reaction is carried out for 2 hours. The suspension is taken out and centrifuged at a speed of 3500 rpm, washed with deionized water 3 times, and then centrifuged again. The solid mixture at the bottom is taken out and dried and ground at 60°C to obtain modified potassium montmorillonite; ethyl orthosilicate, ethanol and deionized water are mixed in a molar ratio of 1:4:4 at 50°C, dilute hydrochloric acid is added dropwise to adjust the pH to 5, and the mixture is stirred to react to form a transparent sol; 5 parts of the transparent sol are taken, 40 parts of the modified potassium montmorillonite are slowly added, the mixture is stirred for 3 hours, the temperature is raised to 60°C, the mixture is allowed to stand for 12 hours, and then the temperature is lowered to room temperature and allowed to stand for 24 hours. After centrifugation, gel microspheres are obtained, which are washed with water 3 times, dried at 60°C, and then heated to 420°C at 2°C / min and kept warm for 3 hours to obtain a porous carrier.

[0023] Furthermore, the negatively charged NH2 -The method comprises the following steps: ultrasonically dispersing 50 parts of the porous carrier into 100 parts of toluene at room temperature, then heating to 80°C, slowly adding 20 parts of (3-methylaminopropyl)trimethoxysilane under stirring, and continuing stirring for 6 hours; cooling to room temperature after the reaction, centrifuging, washing with a large amount of ethanol and water, and drying to obtain a layer with negatively charged NH2 - Amination of porous supports containing groups; Furthermore, the preparation method of the positively charged porous active carrier is as follows: at a temperature of 60°C, chitosan is prepared into 100 parts of a chitosan solution with a concentration of 5% using a 0.05% citric acid aqueous solution, 50 parts of the aminated porous carrier is added to the chitosan solution, and the mixture is stirred for 100 minutes. After the reaction is completed, 10 parts of glucose and 15 parts of sodium alginate are added, and the stirring reaction is continued for 80 minutes. Finally, the mixture is filtered, dried, ground, and filtered through a 60-mesh filter to obtain the positively charged porous active carrier. Take the seed liquid of Chlorella pyrenoidosa, and the density of Chlorella pyrenoidosa is 6×10 6 cfu / g or more, inoculate Chlorella pyrenoidosa into BG11 culture medium with an inoculation volume of 5% to 15% and an initial density of 6×10 6 / mL, cultured at 25 ° C, pH = 6.5, light intensity 2300 lux, 24 hours a day, culture time is 5 days, and the pyrenoid chlorella liquid is obtained, wherein the number of viable bacteria of pyrenoid chlorella is ≥10 9 CFU / g; 20 parts of a positively charged porous active carrier were added to 100 parts of a protein core Chlorella liquid, and the mixture was evenly mixed to obtain a dispersion. After standing for 2 hours, the precipitated part was washed with deionized water and freeze-dried to obtain a microalgae biological soil remediation agent.

[0024] Example 2 A biological microalgae soil remediation agent comprises a porous active carrier and biological microalgae loaded in the porous active carrier, wherein the porous active carrier is a porous carrier obtained by compounding silicon dioxide on montmorillonite, specifically: 50 parts of calcium-based montmorillonite are added to 30 parts of deionized water, an excess of 1 mol / L KCl solution is added, and stirring is continued for 10 hours to allow the Ca 2+ K + Replacement, washed with deionized water until free of Cl -After centrifugation, 50 parts of ethanol are added and stirred evenly to fully disperse the potassium montmorillonite, the temperature is raised to 40°C, 25 parts of octadecyltrimethylammonium chloride are added, the mixture is fully stirred, and the reaction is carried out for 2 hours. The suspension is taken out and centrifuged at a speed of 3500 rpm, washed with deionized water 3 times, and centrifuged again. The solid mixture at the bottom is taken out and dried and ground at 60°C to obtain modified potassium montmorillonite; ethyl orthosilicate, ethanol and deionized water are mixed in a molar ratio of 1:4:4 at 40°C, dilute hydrochloric acid is added dropwise to adjust the pH to 4.5, and the mixture is stirred to react to form a transparent sol; 5 parts of the transparent sol are taken, 40 parts of the modified potassium montmorillonite are slowly added, the mixture is stirred for 3 hours, the temperature is raised to 60°C, the mixture is allowed to stand for 12 hours, and then the temperature is lowered to room temperature and allowed to stand for 24 hours. After centrifugation, gel microspheres are obtained, which are washed with water 3 times, dried at 60°C, and then heated to 480°C at 2°C / min and kept warm for 3 hours to obtain a porous carrier.

[0025] Furthermore, the negatively charged NH2 - The method comprises the following steps: ultrasonically dispersing 50 parts of the porous carrier into 100 parts of toluene at room temperature, then heating to 90°C, slowly adding 30 parts of (3-methylaminopropyl)trimethoxysilane under stirring, and continuing stirring for 6 hours; cooling to room temperature after the reaction, centrifuging, washing with a large amount of ethanol and water, and drying to obtain a layer of negatively charged NH2 - Amination of porous supports containing groups; Furthermore, the preparation method of the positively charged porous active carrier is as follows: at a temperature of 60°C, chitosan is prepared into 100 parts of a chitosan solution with a concentration of 3% using a 0.05% citric acid aqueous solution, 40 parts of the aminated porous carrier is added to the chitosan solution, and the mixture is stirred and reacted for 80 minutes. After the reaction is completed, 15 parts of glucose and 18 parts of sodium alginate are added, and the mixture is stirred and reacted for another 80 minutes. Finally, the mixture is filtered, dried, ground, and filtered through a 60-mesh filter to obtain the positively charged porous active carrier. Take the seed liquid of Chlorella pyrenoidosa, and the density of Chlorella pyrenoidosa is 6×10 6 cfu / g or more, inoculate Chlorella pyrenoidosa into BG11 culture medium with an inoculation volume of 5% to 15% and an initial density of 6×10 6 / mL, cultured at 25 ° C, pH = 6.7, light intensity 2000 lux, 24 hours a day, culture time is 4 days, and the pyrenoid chlorella liquid is obtained, wherein the number of viable bacteria of pyrenoid chlorella is ≥10 9 CFU / g; 30 parts of a positively charged porous active carrier were added to 100 parts of a protein core Chlorella liquid, and the mixture was evenly mixed to obtain a dispersion. After standing for 2 hours, the precipitated part was washed with deionized water and freeze-dried to obtain a microalgae biological soil remediation agent.

[0026] Example 3 A biological microalgae soil remediation agent comprises a porous active carrier and biological microalgae loaded in the porous active carrier, wherein the porous active carrier is a porous carrier obtained by compounding silicon dioxide on montmorillonite, specifically: 50 parts of calcium-based montmorillonite are added to 30 parts of deionized water, an excess of 1 mol / L KCl solution is added, and stirring is continued for 10 hours to allow the Ca 2+ K + Replacement, washed with deionized water until free of Cl - After centrifugation, 50 parts of ethanol are added and stirred evenly to fully disperse the potassium montmorillonite, the temperature is raised to 40°C, 25 parts of octadecyltrimethylammonium chloride are added, the mixture is fully stirred, and the reaction is carried out for 2 hours. The suspension is taken out and centrifuged at a speed of 3500 rpm, washed with deionized water 3 times, and centrifuged again. The solid mixture at the bottom is taken out and dried and ground at 60°C to obtain modified potassium montmorillonite; ethyl orthosilicate, ethanol and deionized water are mixed in a molar ratio of 1:4:4 at 40°C, dilute hydrochloric acid is added dropwise to adjust the pH to 4.5, and the mixture is stirred to react to form a transparent sol; 5 parts of the transparent sol are taken, 40 parts of the modified potassium montmorillonite are slowly added, the mixture is stirred for 3 hours, the temperature is raised to 60°C, the mixture is allowed to stand for 12 hours, and then the temperature is lowered to room temperature and allowed to stand for 24 hours. After centrifugation, gel microspheres are obtained, which are washed with water 3 times, dried at 60°C, and then heated to 450°C at 2°C / min and kept warm for 3 hours to obtain a porous carrier.

[0027] Furthermore, the negatively charged NH2 - The method comprises the following steps: ultrasonically dispersing 50 parts of the porous carrier into 100 parts of toluene at room temperature, then heating to 90°C, slowly adding 20 parts of (3-methylaminopropyl)trimethoxysilane under stirring, and continuing stirring for 6 hours; cooling to room temperature after the reaction, centrifuging, washing with a large amount of ethanol and water, and drying to obtain a layer with negatively charged NH2 - Amination of porous supports containing groups; Furthermore, the preparation method of the positively charged porous active carrier is as follows: at a temperature of 60°C, 150 parts of chitosan solution with a concentration of 5% is prepared using a 0.05% citric acid aqueous solution, 50 parts of the aminated porous carrier is added to the chitosan solution, and the reaction is stirred for 90 minutes. After the reaction is completed, 15 parts of glucose and 20 parts of sodium alginate are added and the stirring reaction is continued for 60 minutes. Finally, the porous active carrier is filtered, dried, ground, and filtered through 60 mesh to obtain a positively charged porous active carrier.

[0028] Take the seed liquid of Chlorella pyrenoidosa, and the density of Chlorella pyrenoidosa is 6×10 6cfu / g or more, inoculate Chlorella pyrenoidosa into BG11 culture medium with an inoculation volume of 5% to 15% and an initial density of 6×10 6 / mL, cultured at 25 ° C, pH = 7.0, light intensity 2500 lux, 24 hours a day, culture time is 4 days, and the pyrenoid chlorella liquid is obtained, wherein the number of viable bacteria of pyrenoid chlorella is ≥10 9 CFU / g; 30 parts of a positively charged porous active carrier were added to 100 parts of a protein core Chlorella liquid, and the mixture was evenly mixed to obtain a dispersion. After standing for 2 hours, the precipitated part was washed with deionized water and freeze-dried to obtain a microalgae biological soil remediation agent. The microalgae biological soil remediation agent and the nutrient solution are evenly mixed in a mass ratio of 8:80; when the culture component in the nutrient solution accounts for 0% by weight, the microalgae biological soil remediation agent is dispersed and diluted; and the obtained dispersion is used for soil drip irrigation.

[0029] Example 4 The microalgae biological soil remediation agent prepared in Example 3 was evenly mixed with the nutrient solution at a mass ratio of 8:80. When the culture component in the nutrient solution was 0.1%, the culture component included sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, citric acid, ammonium ferric citrate, disodium EDTA, sodium carbonate and trace elements, and the weight ratio of sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, citric acid, ammonium ferric citrate, disodium EDTA, sodium carbonate and trace elements was The content of the minerals in the raw materials is (110-130):(3-3.4):(5-8):(2.6-3):(0.45-0.5):(0.45-0.5):(0.05-0.09):(1.3-1.8):(0.04-0.06), and the trace elements include H3BO3, MnCl2·4H2O, ZnSO4·7H2O, Na2MoO4·2H2O, CuSO4·H2O and Co(NO3)2·6H2O. The weight ratio of H3BO3, MnCl2·4H2O, ZnSO4·7H2O, Na2MoO4·2H2O, CuSO4·H2O and Co(NO3)2·6H2O is (2.8-2.9):(1.8-1.9):(0.2-0.23):(0.35-0.4):(0.06-0.08):(0.03-0.06), and the nutrient solution is prepared according to the above ratio; The microalgae biological soil remediation agent is mixed with the nutrient solution, and after uniform mixing, the corresponding container is covered; further, a ventilation gap is left on the covered container; further, the container can be a white container with a certain degree of transparency, and is placed in the corresponding container for fermentation for 3 days at a fermentation temperature of 28° C. During the fermentation process, the container is opened at least once a day for stirring, and then the container is continued to be covered and fermented. After the fermentation is completed, the corresponding container is opened to obtain a microalgae expansion solution; the microalgae expansion solution is applied to the soil for soil remediation.

[0030] Comparative Example 1: The cultured Chlorella pyrenoidosa liquid was directly used as a microalgae biological soil remediation agent, and the rest was the same as in Example 3; Comparative Example 2: The positively charged porous active carrier was directly replaced by an equal amount of calcium-montmorillonite, and the other conditions were the same as those in Example 3. Comparative Example 3: Directly using negatively charged NH2 - The aminated porous carrier of the group replaces the positively charged porous active carrier in equal amounts, and the rest is the same as in Example 3; Comparative Example 4: the active microalgae nutrient repair solution product provided in Example 8 of the Chinese patent publication No. CN109401977B; Comparative Example 5: Common nitrogen and phosphorus fertilizers were used to fertilize the sweet potato control plot three times; In comparative example 6, the sea rice control area was fertilized three times using a special fertilizer for rice.

[0031] Test data and result analysis 1. Storage activity The survival rate of microalgae active cells in the microalgae biological soil remediation agent was tested. The microalgae remediation products prepared in Examples 1-3 and Comparative Examples 1-4 were stored at room temperature in a dark environment for 10 days, 1 month, 3 months, and 6 months. Seven active microalgae remediation products were examined under a microscope. The survival rate of the microalgae was calculated as the ratio of the number of microalgae active cells at the time of microscopic examination to the number of microalgae active cells at the initial time. The products were shaken well before microscopic examination. The test results are shown in Table 1.

[0032] The microalgae restoration products prepared in Examples 1-3 and Comparative Examples 1-4 were stored in a dark environment at 50° C. for 1 day, 10 days, and 15 days. Seven samples of active microalgae restoration products were examined under a microscope. The test results are shown in Table 1.

[0033] Table 1 Results of viability test of microalgae cells at room temperature

[0034] Table 2 Results of microalgae cell survival rate test under high temperature

[0035] As shown in Table 1, at room temperature, the active microalgae nutrient remediation solutions prepared in Examples 1-3 and Comparative Examples 1-4 maintained a survival rate of over 92% for Chlorella pyrenoidosa after six months, demonstrating the excellent compatibility and symbiosis of Chlorella pyrenoidosa, resulting in a long shelf life for these microalgae-based soil remediation products. Furthermore, when the temperature was raised to 50°C, the microalgae bioremediation agents in Examples 1-3 maintained a survival rate of approximately 40% after 15 days of storage, while the microalgae in Comparative Examples 1-4 barely survived. This is primarily because Chlorella pyrenoidosa is highly sensitive to temperature and enters a dormant state when exposed to high temperatures. Continued exposure quickly deactivates the cells. The microalgae bioremediation agents in Examples 1-3, protected by a double layer of montmorillonite and sodium alginate, allow the Chlorella pyrenoidosa to remain dormant within the micropores. However, prolonged exposure to high temperatures severely damages the outer cell membrane of the Chlorella pyrenoidosa, leading to its inactivation. However, the survival rate of the microalgae bioremediation agents in Comparative Examples 1-4 is significantly improved compared to those in Comparative Examples 1-4.

[0036] 2. Activity detection in soil Contaminated acidic soil with a pH of approximately 5.0 was evenly spread into 20 cm square test plots 1-4. Normally cultivated soil was collected from the control test plot. The microalgae bioremediation product prepared in each example / comparative example was added to one of the soil groups and mixed evenly, with the mass of the microbial preparation added to each soil accounting for 1% of the soil mass. Square plot 1: light intensity 2000, pH around 5.0, temperature 30°C; Square plot 2: light intensity 5000 (12 hours light, 12 hours dark), pH around 5.0, temperature 30°C; Square plot 3: light intensity 500 (12 hours light, 12 hours dark), pH around 5.0, temperature 30°C; Square plot 4: light intensity 500 (12 hours light, 12 hours dark), pH around 5.0, surface temperature 50°C, simulating high summer temperatures, 8 hours of high temperature 50°C, 16 hours of low temperature 30°C; Control group test site: light intensity 2000 (12 hours light, 12 hours dark), pH value around 7.0, temperature 30°C; Immediately after fertilization, take 5 portions of 1g soil sample from the surface, 5 portions of 1g soil sample from a depth of 5cm, and 5 portions of 1g soil sample from a depth of 20cm; 48 hours after fertilization, 5 portions of 1 g of surface soil sample, 5 portions of 1 g of middle layer soil sample, and 5 portions of 1 g of soil sample at a depth of 20 cm were collected from each test plot; After fertilization and 48 hours after fertilization, each soil sample was added to 10 ml of sterile phosphate buffer, vortexed for 5 minutes, and allowed to settle. Algal cells were enriched using Percoll gradient centrifugation to remove soil impurities. The algal cells were then filtered through a 10 μm sterile filter membrane to remove large particles. Finally, the algal cells were mixed with the dye fluorescein diacetate and incubated in the dark for 15 minutes. The number of viable cells was counted using a fluorescence microscope. See Table 3-5. Table 3 Amount of living cells in surface soil samples

[0037] As shown in Table 3, when the surface layer is exposed to sufficient light, Examples 1-3 can overcome the inhibition caused by low pH, rapidly reproduce, and maintain sufficient activity. When light is insufficient, they can rapidly activate the heterogeneous matrix, allowing them to maintain sufficient activity while slowing reproduction. Furthermore, when exposed to high temperatures, they can enter a dormant period while maintaining activity. This is because Chlorella pyrenoidosa has a rapid growth rate and strong environmental tolerance. Under nitrogen-rich conditions, it can synthesize abundant nitrogen-containing compounds (such as chlorophyll and protein) within its cells. Examples 1-3 not only protect Chlorella pyrenoidosa but also provide nutrients, enabling it to grow rapidly in both low and high light conditions, maintain vitality for a long time, and maintain a healthy, sustainable microecosystem. Furthermore, research has shown that protonation or deprotonation reactions at the edge sites of montmorillonite and proton exchange reactions at the basal sites give montmorillonite the ability to buffer pH. Over 60 hours, the soil pH showed an upward trend, while the pH of the microalgae-montmorillonite composite reaction system remained relatively stable. This indicates that the addition of modified montmorillonite mitigated pH fluctuations during microalgae growth, eliminating the negative impact of pH reduction on microalgae growth and reproduction. However, in Comparative Examples 1 and 4, the microalgae lacked a protective layer and a sustainable nutrient source, resulting in rapid death and inactivation in the absence of sufficient light. Furthermore, in Comparative Examples 2 and 3, insufficient nutrients and relatively weak protective effects slowed growth, leading to rapid inactivation of some microalgae due to the harsh living environment.

[0038] Table 4 The number of living cells in soil samples at a depth of 5 cm

[0039] Table 5 The number of living cells in soil samples at a depth of 20 cm

[0040] As can be seen from Table 3-5, the survival rate of the number of live bacteria in the microalgae biological soil remediation agent in Example 1-3 is high. Under high temperature and low light conditions, the heterotrophic mode can be activated, and the microalgae can slowly reproduce and grow, while the number of live bacteria in Comparative Example 1-4 can maintain activity is relatively small. This is because although the protein nucleus Chlorella can tolerate an acidic environment for a period of time, it does not reproduce in an acidic and high temperature environment, and it will continue to inactivate in an acidic and high temperature environment for more than 10 hours; and the inactivated bacteria are also easy to die during storage, and the number of live bacteria continues to decrease over time. The microalgae in the surface soil have sufficient light, so their growth and reproduction speed will be faster than that in the middle layer and 20cm. After the microalgae biological soil remediation agent of Example 1-3 is applied to the soil in the middle layer, because it is compounded with silica, the light intensity of the remediation agent is faster than that of the remediation agent without compound silica in Comparative Examples 2 and 3, and the growth rate is fast, mainly because its light transmittance is good, so that the light energy passing through the soil gap can be fully utilized. As the soil depth continues to increase, its utilization rate of light will become lower and lower, causing the mixotrophic mode to be transformed into a heterotrophic mode, which further reduces the growth and reproduction rate and has basically nothing to do with light intensity.

[0041] 3. Heavy metal ion adsorption capacity of microalgae The degradation ability of heavy metals in soil was tested using microalgae biological soil remediation products: contaminated soil around the smelting waste slag pile was taken, air-dried, crushed, passed through a 20-mesh sieve, thoroughly mixed and placed in a high-pressure sterilizer, sterilized at 110°C for 100 minutes, and cooled. The sterilized soil was divided into 24 parts on average and placed in disinfected containers respectively, and the 24 parts of sterilized soil were divided into 6 groups, with 4 parts in each group. The microalgae biological soil remediation products prepared in Examples 1 to 3 and Comparative Examples 1 to 4 were taken, and the microalgae biological soil remediation products prepared in each Example / Comparative Example were added to one of the groups of soil and mixed evenly, wherein the mass of the microbial preparation added to each part of the soil accounted for 0.5% of the mass of the soil. Three months later, the exchangeable removal rate of heavy metals in the treated soil was tested, and the average value of 3 parts in each group was taken. The results are shown in Table 6. Table 6 Removal rate of heavy metals in soil

[0042] As shown in Table 6, Examples 1-3 all achieved relatively high removal rates for various heavy metals in the soil, while the microalgae in Comparative Examples 1-4 also achieved relatively good removal rates for heavy metals. This is because the microalgae's ability to adsorb heavy metal ions can be attributed to the numerous functional groups on their cell surfaces, such as carboxyl, sulfonic acid, thioether, and phosphogroups. The extensive adsorption of heavy metal ions on the microalgae cell surface can also immobilize the heavy metal ions on the cell surface, hindering their intracellular entry and, to a certain extent, maintaining the activity of the microalgae cells. Furthermore, photosynthetic microorganisms such as microalgae can preferentially produce polypeptides that bind heavy metal ions, which is highly beneficial for the biosorption of heavy metal ions. The microalgae bioremediation agents in Examples 1-3 use a positively charged porous active carrier as the carrier. The interaction between the montmorillonite and the microalgae in the system provides more binding sites for heavy metals, which can promote the loading of more heavy metals onto the system surface. However, under sufficient sunlight, Comparative Example 2 exhibited a low heavy metal removal rate because it used unmodified montmorillonite, and the opaque montmorillonite may have hindered photosynthesis, resulting in a weak growth and reproduction capacity of the microalgae in the soil. While the addition of silica to Comparative Example 3 improves light transmittance and results in a higher heavy metal removal rate than Comparative Example 2, the negative charge of the montmorillonite in the formulation repels the negative charge of the microalgae, thereby affecting their growth and reproduction. The microalgae bioremediation agents in Examples 1-3, on the other hand, fully utilize photosynthesis to achieve an autotrophic mode while also enabling a mixotrophic mode. Furthermore, the synergistic effects of the montmorillonite and chitosan significantly enhance their heavy metal removal rates.

[0043] 4. Monitoring of crop growth status The test site was Yingdie Village, Kelu Town, Leizhou City, and Zhanzishu No. 3 was used as the test object to determine the effects of the fertilizers of Example 3 and Comparative Example 5 on Zhanzishu No. 3. The soil condition of the demonstration group after fertilization with the microalgae biological soil remediation agent prepared in Example 3 for 140 days was shown in the figure. Figure 1 ; Use the fertilizer of comparative example 5 to the soil condition diagram of the control group fertilizing 140 days later, see Figure 1 ;from Figure 1Comparison of soil conditions shows that after the microalgae biological soil remediation agent of Example 3 was applied to the demonstration group, the soil condition improved significantly, changing from a hardened state to a loose and porous state. This is because the microalgae participate in improving soil structure mainly through the aggregation of clay minerals and the bonding effect of colloids. Microorganisms aggregate fine soil particles into clusters through substances such as extracellular polysaccharides and adhesive proteins in colloidal particles. This clustered structure increases the porosity of the soil, providing better ventilation and drainage conditions for plant root growth. At the same time, the clustered structure can also enhance the soil's resistance to wind and water erosion, maintain soil stability, and significantly increase the quality and product of crops. The demonstration group fertilized with Example 3 had an average yield of 2592.8 kg per mu, 851.5 kg more than the control group fertilized with Comparative Example 5. The demonstration area was fertilized with 3 kg of microalgae biological soil remediation agent per mu three times during the seedling stage, once every 7 days. The control area was fertilized with 3 kg of ordinary nitrogen and phosphorus fertilizer according to Comparative Example 5 three times, once every 7 days. Figure 2 This is a comparison of the root systems of sweet potatoes on the 8th day after fertilization 3 times. Figure 2 It can be seen that the root system of Zhan Zishu No. 3 in the demonstration group is well developed, and each root system produces 3-5 more fruits than the control group. Figure 3 Comparison of the root system of sweet potatoes on the 36th day, from Figure 3 It can be seen that the root system of Zhan Zishu No. 3 in the demonstration group is well developed, and the fruits produced by each root system have a more significant growth momentum than the control group, and the fruits are not only more but also larger. Figure 4 This is the harvest photo of Zhan Zishu No. 3. It can also be seen that the fruits of the demonstration group are large and numerous. Figure 5 These are the changes in Zhan Zishu No. 3 during its growth process, which reflect that after using microalgae biological soil remediation agent, the soil condition has improved, the organic matter content has increased, and the crops have grown well, significantly increasing its per-acre yield.

[0044] At the Xie Zong base in Gangmen, a demonstration group using the microalgae propagation solution prepared in Example 4 for fertilization of Pushu 32 achieved a yield of 4732.64 kg per mu. This represents a 904.76 kg increase, or 23.64%, compared to the control group using the cultured Chlorella pyrenoidosa solution prepared in Comparative Example 1 as a microalgae bioremediation agent. All data were measured using standardized ridges: 3 meters wide and 1.2 meters wide.

[0045] The demonstration group planting "Silky Sweet Potato" and fertilizing with the microalgae propagation solution prepared in Example 4 had an annual yield of 2999.09 kg per mu, an increase of 224.35 kg. The yield was measured at three points with two ridges of 5 meters.

[0046] The test was conducted in Suixi County, Zhanjiang, Guangdong Province, with sea rice as the test subject. The effects of different microalgae fertilizers in Examples 1-3, Comparative Examples 1-4, and Comparative Example 6 on soil fertility were determined. The soil organic matter content in the topsoil layer (0-20 cm) of the test site was 8.56 g / kg, the moisture content was 10.7%, and the heavy metal content was: 62.7 mg / kg total Cr, 38.5 mg / kg total Cu, and 6.5 mg / kg total As. A control group was planted with sea rice using conventional local methods (three applications of fertilization: base fertilizer and two topdressings, with base fertilizer being 50 kg / mu of rice-specific fertilizer (15-21-9)). The sea rice planting management of the experimental group was the same as that of the control group, except that when fertilization was required at each growth period of the sea rice (corresponding to the 3 fertilization periods of the control group), 4 kg / mu of microalgae biological soil remediation products of Examples 1-3, Comparative Examples 1-4 and 6 were applied respectively, and fertilization was applied 3 times; when the sea rice in the experimental group was fertilized with Example 3 and Comparative Example 6 on the 56th day of growth, the sea rice was compared. Figure 6 It can be seen that the sea rice obtained by fertilization in Example 3 grew faster and had more and whiter roots than the conventional rice in Comparative Example 6. At harvest, the organic matter content, moisture content, and heavy metal content of the topsoil layer (0-20 cm) of the experimental and control groups were measured. The results are shown in Table 7.

[0047] Table 7 Effects of different groups on soil fertility and heavy metal content

[0048] As shown in Table 7, microalgae can be used as soil conditioners. In addition to removing heavy metals, they can also significantly improve soil structure and increase soil fertility. Microalgae can improve soil water retention and air permeability, providing a better growth environment for plants. This not only helps to improve crop yield and quality, but also reduces environmental problems such as soil erosion and water loss. Microalgae can not only survive and reproduce in the soil and improve the soil biological structure, but after decomposition, they can also act as natural biostimulants to promote plant growth. Applying microalgae to the soil can not only provide a nitrogen source, but also increase the content of other elements in the soil and improve soil fertility. Biological soil crust can strongly affect the content of organic matter in the 0-5cm soil layer on the surface, and can make the organic matter content of this layer significantly higher than that of the area without crust coverage.

[0049] The above description is only used to illustrate the technical solution of the present invention and is not intended to limit it. Any modifications and changes made to the technical solution of the present invention by ordinary persons in the art shall still fall within the scope of the present invention as long as they do not depart from the overall concept of the present invention.

Claims

1. A microalgae biological soil remediation agent, characterized by: The biological microalgae soil remediation agent includes a porous active carrier and biological microalgae loaded in the porous active carrier. The porous active carrier is obtained by compounding silicon dioxide on montmorillonite, and the porous carrier is modified to obtain a negatively charged NH2 - The porous carrier is aminated with chitosan cations and finally loaded with chitosan cations to become a porous active carrier with positive charge; The positively charged porous active carrier is added to the protein core Chlorella liquid, mixed evenly to obtain a dispersion, and allowed to stand for 1 to 2 hours, and the precipitated part is washed with deionized water and freeze-dried to obtain a microalgae biological soil remediation agent; The biological microalgae include pyrenoid chlorella liquid, and the density of pyrenoid chlorella is 6×10 6 cfu / g or above.

2. The microalgae biological soil remediation agent according to claim 1, characterized in that The porous carrier is a porous carrier obtained by compounding silicon dioxide on montmorillonite. Specifically, calcium-based montmorillonite is added to deionized water, an excess of 1 mol / L KCl solution is added, and stirring is continued for 10 to 12 hours to allow Ca 2+ K + Replacement, washed with deionized water until free of Cl - After centrifugation, ethanol was added and stirred evenly to fully disperse the potassium montmorillonite. The temperature was raised to 40° C., octadecyltrimethylammonium chloride was added, stirred thoroughly, and reacted for 1 to 2 hours. The suspension was removed and centrifuged at 3500 rpm, washed 2 to 3 times with deionized water, and centrifuged again. The solid mixture at the bottom was removed and dried and ground at 60° C. to obtain modified potassium montmorillonite. The mass ratio of the octadecyltrimethylammonium chloride to the potassium montmorillonite was 1:

2. TEOS, ethanol, and deionized water are mixed in a molar ratio of 1:4:4 at 40-50°C, diluted hydrochloric acid is added dropwise to adjust the pH to 4-5, and the mixture is stirred to form a transparent sol; modified potassium montmorillonite is slowly added, stirred for 2-3 hours, heated to 60°C, allowed to stand for 12 hours, then cooled to room temperature and allowed to stand for 24 hours, centrifuged and filtered to obtain gel microspheres, which are washed with water 2-3 times, dried at 60°C, heated to 400-500°C at 2°C / min, and kept warm for 3 hours to obtain a porous carrier; the mass ratio of the transparent sol to potassium montmorillonite is 1:

8.

3. The microalgae biological soil remediation agent according to claim 1, characterized in that There is negative charge NH2 between the layers - The method comprises the following steps: ultrasonically dispersing the porous carrier into toluene 2 to 3 times the weight of the porous carrier at room temperature, then heating the porous carrier to 80 to 90°C, slowly adding (3-methylaminopropyl)trimethoxysilane under stirring, and continuing stirring for 5 to 6 hours; cooling the porous carrier to room temperature after the reaction, centrifuging the porous carrier, washing the porous carrier with a large amount of ethanol and water, and drying the porous carrier to obtain a layer of negatively charged NH2 - The aminated porous carrier comprises a plurality of groups; wherein the mass ratio of (3-methylaminopropyl)trimethoxysilane to modified montmorillonite is 2 to 3:

5.

4. The microalgae biological soil remediation agent according to claim 1, characterized in that The preparation method of the positively charged porous active carrier is specifically as follows: at a temperature of 50-60°C, chitosan is prepared into a chitosan solution with a concentration of 3-5% using a 0.05% citric acid aqueous solution, the aminated porous carrier is added to the chitosan solution, and the reaction is stirred for 80-100 minutes. After the reaction is completed, glucose and sodium alginate are added and the stirring reaction is continued for 50-80 minutes. Finally, the mixture is filtered, dried, ground, and filtered through 60 mesh to obtain the positively charged porous active carrier. The mass ratio of the chitosan, sodium alginate, glucose, and aminated porous carrier is 3-8:15-20:10-15:40-50.

5. The microalgae biological soil remediation agent according to claim 1, characterized in that Chlorella pyrenoidosa was inoculated into BG11 culture medium at an inoculation rate of 5% to 15% and an initial density of 6 × 10 6 The culture is carried out at a temperature of 20-25°C, pH=6.5-7.1, light intensity of 2000-3000 lux, and 24 hours of light per day for 3-5 days to obtain a pyrenoid Chlorella liquid, wherein the number of viable bacteria of the pyrenoid Chlorella is ≥10 9 CFU / g; the mass ratio of the positively charged porous active carrier to the protein core Chlorella liquid is 2 to 3:

10.

6. A soil remediation method based on the biological soil remediation agent according to claim 1, comprising uniformly mixing the microalgae biological soil remediation agent and the nutrient solution in a mass ratio of 8 to 10:80; When the culture component accounts for 0% by weight in the nutrient solution, the method comprises the following steps: dispersing and diluting the microalgae biological soil remediation agent; drip irrigation of the obtained dispersion; When the culture component accounts for more than 0% by weight of the nutrient solution, the method includes the following steps: mixing a microalgae biological soil remediation agent with the nutrient solution, and covering the corresponding container after uniform mixing; further, the covered container is provided with a ventilation gap; further, the container can be a white container with a certain degree of transparency, placed in the corresponding container for fermentation for 2 to 3 days at a fermentation temperature of 25 to 35° C. During the fermentation process, the container is opened at least once a day for stirring, and then the container is continuously covered and fermented. After the fermentation is completed, the corresponding container is opened to obtain a microalgae expansion solution; and the microalgae expansion solution is applied to the soil to perform soil remediation.

7. The soil remediation method using the biological soil remediation agent according to claim 6, characterized in that: The nutrient solution comprises water and a culture component accounting for 0.1-0.18% by weight; the culture component comprises sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, citric acid, ammonium ferric citrate, disodium EDTA, sodium carbonate and trace elements, and the weight ratio of sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, citric acid, ammonium ferric citrate, disodium EDTA, sodium carbonate and trace elements is (110-130): (3-3.4): (5-8): (2.6-3): (0.45-0.5): (0.45-0.5): (0.05-0.09): (1.3-1.8): (0.04-0.06).

8. The soil remediation method using the biological soil remediation agent according to claim 7, characterized in that: The trace elements include H3BO3, MnCl2·4H2O, ZnSO4·7H2O, Na2MoO4·2H2O, CuSO4·H2O and Co(NO3)2·6H2O, and the weight ratio of H3BO3, MnCl2·4H2O, ZnSO4·7H2O, Na2MoO4·2H2O, CuSO4·H2O and Co(NO3)2·6H2O is (2.8~2.9):(1.8~1.9):(0.2~0.23):(0.35~0.4):(0.06~0.08):(0.03~0.06).

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