Microalgae bioremediation agent and preparation method thereof

Abstract

The application 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, and the prepared microalgae biological soil remediation agent takes a porous active carrier with positive charges as a matrix.The porous active carrier is obtained by compounding silicon dioxide on montmorillonite, and the porous active carrier is modified to obtain an amine group of a negative charge NH2 ‑ between layers, and finally loaded with chitosan cations to become a porous active carrier with positive charges, which is loaded with a large amount of Chlorella pyrenoidosa with reduced environmental sensitivity, so that it can survive and reproduce in harsh soil environments, can significantly improve soil fertility, improve crop yield and quality, and also has the effect of reducing soil heavy metal pollution; can also increase the diversity of soil microbial communities, improve soil aggregate structure, and improve the soil physical and chemical environment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of soil remediation, and particularly relates to a preparation method of a microalgae biological soil remediation agent and application thereof. BACKGROUND

[0002] The average content of organic matter in soil is less than 1%, and it takes 100 years to naturally increase 1%. Overexploitation causes vegetation damage and soil erosion, and ecological restoration may take thousands of years or more. The surface layer of soil lacks organic matter, the nutrient ratio is out of balance, the soil lacks capillary pore space, and the soil has poor aeration and water permeability, poor microbial activity, and poor water and fertilizer retention and air permeability, which makes it difficult to meet the actual growth needs of crops. In addition, industrial pollution exacerbates the destruction of soil organic matter.

[0003] Without organic matter and microorganisms, the soil granule structure is destroyed, and even if more nitrogen, phosphorus, and potassium are applied, it is of no avail. Although there is a lot of nitrogen, phosphorus, and potassium in the soil, it cannot be effectively absorbed and utilized by crops. This is because most of the nutrients applied to the soil are lost, and the actual utilization rate is only 30% on average. Most of the nutrients flow into rivers and lakes with farmland drainage, not only causing environmental pollution but also endangering human safety and health. In addition, the soil still shows a lack of nutrients, fertility, and activity, and may also have problems such as soil acidification, hardening, trace element deficiency, and excessive heavy metals. The root exudates and decomposed substances of crops (such as p-hydroxybenzoic acid PA) can inhibit the growth and activity of continuous cropping plant roots, and have the strongest autotoxicity, resulting in a decrease in the content of beneficial microorganisms in the soil, an imbalance of microorganisms in the soil, and continuous cropping obstacles in the growth process of crops. In order to effectively ensure the potential fertility of the soil, more and more regions and countries add a large amount of beneficial microorganisms to the soil to better ensure the activity of microorganisms in the soil and comprehensively improve the nutritional conditions of the soil. However, there are few reports on the use of algal active cell biofertilizer.

[0004] A soil remediation method is disclosed in Chinese Patent Publication No. CN111215442B, which provides a soil remediation method. The soil to be repaired is covered with water, and then the covered soil is dried. A signal agent and a recovery agent are then applied. The recovery agent includes deep-sea fish protein, soybean soft phospholipid, alfalfa powder, aloe vera, and organic seaweed. The signal agent includes shellfish, grains, and spherical algae. The soil to be repaired is first subjected to large water pressure to kill microbial hibernation eggs, and then the signal agent is used to quickly awaken and activate underground indigenous microorganisms, allowing them to work together to create an environment conducive to microbial survival through the use of a recovery agent, improve aggregate structure, purify and transport nutrients, and supply crops for growth. The crop root system secretes exudates to support microbial survival, forming a sustainable and benign underground microecosystem. Although the invention mentions the formation of a sustainable and benign underground microecosystem, it does not provide information on how to maintain it, and does not address the problem of the activity of spherical algae and fermented products in outdoor high-temperature harsh soil.

[0005] Chinese Patent Publication No. CN109401977B discloses an active microalgae nutrient repair liquid and its preparation method. It discloses the use of green algae protein nuclear Chlorella vulgaris and nitrogen-fixing blue-green algae water bloom fishy algae to obtain two kinds of algae liquid. The two kinds of algae liquid are mixed to obtain an active microalgae nutrient repair liquid. The active microalgae nutrient repair liquid provided by the patent has the effects of repairing soil, supplementing natural nitrogen, activating soil trace elements, improving plant stress resistance, preventing pests and diseases, and improving the quality and yield of agricultural products. However, the patent does not address the duration of the activity of these microalgae during soil remediation and the problem of how microalgae can maintain their activity for a long time and continue to repair the soil when they are in the deep soil with little light and energy during soil remediation.

[0006] Microalgae bioremediation method can use microalgae to enrich and fix heavy metals in soil through biological accumulation, cell surface adsorption and biomineralization. Microalgae are autotrophic organisms with strong carbon fixation and nitrogen fixation capacity, which is beneficial to the ecological restoration of heavy metal contaminated soil. The biological accumulation process depends on the cell metabolism of microalgae. Heavy metal ions transport or diffuse through the cell membrane through special carrier proteins, and then combine with intracellular organelles or compounds, thereby being fixed, which is slow and irreversible. However, in the process of soil remediation, especially in the deep soil (10 cm below), microalgae will be inactivated due to lack of light and energy. In the surface layer of soil, microalgae cannot maintain activity for a long time due to high temperature. Therefore, how to maximize the use of microalgae to repair soil not only provides a large amount of organic matter, but also better ensures the activity of microorganisms in soil, and ensures that microalgae have strong environmental tolerance and grow fast in soil, forming a sustainable and benign underground microecosystem is the difficulty at present. SUMMARY

[0007] In order to solve the above problems, the purpose of the present application is to provide a preparation method of microalgae biological soil repair agent, so as to solve one or more technical problems existing in the prior art, and at least provide a beneficial choice or create conditions. The purpose of the present application is to provide a preparation method of biological soil repair agent, which is used for repairing soil, can increase the diversity of soil microbial community, improve the soil aggregate structure, and improve the soil physical and chemical environment.

[0008] One purpose of the present application is to provide a biological microalgae soil repair agent, which comprises a porous active carrier and biological microalgae loaded in the porous active carrier, wherein the biological microalgae comprises Chlorella pyrenoidosa liquid, and the density of Chlorella pyrenoidosa is 6x10 6 cfu / g or more; the porous active carrier is a porous carrier obtained by compounding silicon dioxide on montmorillonite, and the porous carrier is modified to obtain an amine group of a negative charge NH2 - group between layers, and finally loaded with chitosan cation to become a positively charged porous active carrier.

[0009] 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 stirring is continued for 10-12 hours, so that Ca 2+ is replaced by K + , and washed with deionized water until Cl -After centrifugal separation, ethanol is added and stirred evenly, the potassium-based montmorillonite is fully dispersed, the temperature is raised to 40℃, octadecyl trimethyl ammonium chloride is added, and fully stirred, and the suspension is centrifugally separated at 3500 rpm, washed with deionized water for 2-3 times, centrifugally separated again, and the solid mixture at the bottom is taken out and dried at 60℃ to obtain the modified potassium-based montmorillonite; the mass ratio of the octadecyl trimethyl ammonium chloride to the potassium-based montmorillonite is 1:2.

[0010] In K + The concentration is high and the hydrated ion radius is smaller than Ca 2+ Therefore, the stirring time is prolonged, the calcium-based montmorillonite is replaced by the potassium-based montmorillonite, the potassium-based montmorillonite is modified by the octadecyl trimethyl ammonium chloride, the interlayer spacing of the potassium-based montmorillonite is expanded, and the problem of swelling of the montmorillonite in water is significantly reduced.

[0011] Ethyl silicate, ethanol and deionized water are mixed at a molar ratio of 1:4:4 at 40-50℃, and dilute hydrochloric acid is added dropwise to pH 4-5, and stirred to form a transparent sol; the modified potassium-based montmorillonite is slowly added, stirred for 2-3 hours, the temperature is raised to 60℃, and the mixture is left to stand for 12 hours, then cooled to room temperature and left to stand for 24 hours, centrifuged and filtered to obtain a gel microsphere, washed with water for 2-3 times, dried at 60℃, and then heated to 400-500℃ at a rate of 2℃ / min to obtain a porous carrier; the mass ratio of the transparent sol to the potassium-based montmorillonite is 1:8.

[0012] Further, the porous carrier is modified to obtain an amine-modified porous carrier with negative charge NH2 - group in the interlayer, specifically:

[0013] The porous carrier is ultrasonically dispersed in toluene at room temperature, the weight of the toluene is 2-3 times of the weight of the porous carrier, then the temperature is raised to 80-90℃, (3-methylaminopropyl) trimethoxysilane is slowly added under stirring, and the stirring is continued for 5-6 hours; after the reaction is completed, the temperature is cooled to room temperature, centrifugally separated, and washed with a large amount of ethanol and water, and dried to obtain an amine-modified porous carrier with negative charge NH2 - group in the interlayer; the mass ratio of the (3-methylaminopropyl) trimethoxysilane to the modified montmorillonite is 2-3:5;

[0014] Further, a positively charged porous active carrier is prepared, specifically:

[0015] The chitosan is prepared into a chitosan solution with a concentration of 3-5% by using 0.05% citric acid aqueous solution at a temperature of 50-60 ℃, the amine group porous carrier is added into the chitosan solution, and stirring reaction is carried out for 80-100 minutes, then glucose and sodium alginate are added to continue stirring reaction for 50-80 minutes, finally, the positively charged porous active carrier is obtained by filtering, drying, grinding and filtering of 60 meshes after the preparation, and the mass ratio of the chitosan, the sodium alginate, the glucose and the amine group porous carrier is 3-8:15-20:10-15:40-50.

[0016] Finally, the microalgae biological soil remediation agent is prepared, specifically as follows:

[0017] The Chlorella pyrenoidosa is inoculated into the BG11 culture solution with an inoculation amount of 5%-15% and an initial density of 6x10 6 The Chlorella pyrenoidosa is cultured at a temperature of 20-25 ℃, pH=6.5-7.1, light intensity of 2000-3000 lux, and 24 hours of light per day, and the culture time is 3-5 days, so that the Chlorella pyrenoidosa solution is obtained, wherein the viable cell count of the Chlorella pyrenoidosa is ≥10 9 CFU / g.

[0018] The positively charged porous active carrier is added into the Chlorella pyrenoidosa solution to obtain a dispersion liquid, and after standing for 1-2 hours, the precipitated part is washed with deionized water, and the microalgae biological soil remediation agent is obtained by freeze-drying; wherein the mass ratio of the positively charged porous active carrier to the Chlorella pyrenoidosa solution is 2-3:10.

[0019] The Chlorella pyrenoidosa belongs to green algae, has strong photosynthesis, and has extraordinary ability to absorb solar energy. As long as there is a visible light source, the Chlorella pyrenoidosa can perform photosynthesis. The Chlorella pyrenoidosa has extremely strong reproductive ability and reproduces by asexual reproduction. A single cell can release 4 new daughter cells each time. The Chlorella pyrenoidosa can not only perform autotrophy by light energy, but also perform heterotrophy and mixotrophic growth. Compared with the photosynthetic autotrophy mode, the heterotrophic culture solves the problem of insufficient light and can achieve higher production efficiency, reduces external pollution, and ensures better product quality of the microalgae. The heterotrophic culture can realize precise control of the growth conditions and is more conducive to inducing the efficient production of specific metabolites. However, when the microalgae biological soil remediation agent is applied to the soil, especially the acidic and barren soil, the microalgae will not only face the problem of insufficient light intensity to start the photosynthetic autotrophy mode, but also cannot obtain external energy support from the surrounding environment. More importantly, the microalgae will face the problem of high surface temperature and harsh external environment, which will quickly lead to inactivation of the microalgae.

[0020] Soil physical and chemical properties include soil structure and soil pH value. Soil structure is actually soil aggregates formed by different arrangement of soil particles. Different arrangement often forms different soil structure. Long-term overuse of chemical fertilizer reduces soil microorganisms and causes serious soil compaction. Soil compaction changes soil properties and causes poor soil porosity, which further affects soil fertility and tillage capacity. Soil acidification refers to the increase of hydrogen ions in soil and the decrease of pH value, which makes soil strongly acidic or extremely acidic. Substances that cause soil acidification include acid rain caused by air pollution, organic acids produced by decomposition of soil organic matter, carbonic acid produced by metabolism of soil microorganisms and plant roots, etc. The use of microalgae liquid fertilizer can improve soil aggregate structure, enhance soil physical properties and reduce soil particle loss. Under certain conditions, it can also participate in the formation of humus. On the one hand, microalgae photosynthesis consumes a large amount of carbon sources such as carbon dioxide. Organic acids such as carbonic acid, acetic acid, succinic acid and citric acid produced by microbial decomposition can be used as carbon sources for microalgae to synthesize organic matter. However, before soil remediation, the soil lacks organic matter and is seriously compacted, so that the applied microalgae biological soil remediation agent cannot survive in such soil environment and quickly loses activity. The high surface temperature and poor soil porosity in the shallow layer of the compacted soil make it difficult for light to reach the intensity required for microalgae to start autotrophy. Therefore, a certain culture environment needs to be provided to enable the applied microalgae to overcome the actual situation of the soil, so that the microalgae can autotrophically and self-sufficiently grow in harsh soil environment, achieve rapid growth and have long-lasting activity.

[0021] Studies have shown that there is a negative charge NH2 -The protonation or deprotonation reaction of the amine group of the porous carrier and the proton exchange reaction of the edge site of the montmorillonite endow the ability of buffering pH, so that the protein core chlorella liquid can survive and reproduce in low or high pH environment, and the sensitivity of the protein core chlorella liquid to the environment is reduced. The montmorillonite is combined with silicon dioxide, and the light transmittance is improved, so that a large amount of light can be obtained in the surface soil, so as to start the photosynthetic autotrophic mode, and in the deep soil, because the microalgae are loaded in the porous active carrier, the energy adsorbed and carried by the carrier can meet the heterotrophic and mixotrophic growth, so that the microalgae can maintain higher activity and form a sustainable benign underground microecosystem. Moreover, the pH can be adjusted to be close to neutral during the reproduction and growth of the microalgae, which can resist the process of soil acidification. On the other hand, through microscope observation, the microalgae liquid fertilizer improves the soil aggregate structure and increases the content of soil surface chlorophyll. The soil aggregate structure determines the plant root planting, water and nutrient uptake, and is the main carrier and embodiment of soil respiration rate, water holding capacity and the like. The sugar substances produced by the microalgae are combined with plant mucus, mineral embryos and organic colloids to improve the soil aggregate structure. Therefore, the protein core chlorella and its metabolites are applied to the soil, and the algal cells can be planted and grown in the soil and plant roots, and oxygen is produced during the reproduction process to provide respiration for the roots, increase the soil porosity and prevent soil compaction. The advantages and disadvantages of the soil aggregate structure are directly related to the growth of plants and the sustainable development of soil health. The soil with too tight aggregate structure is easy to cause hypoxia in the lower layer, forming a heterotrophic environment, so that the soil properties and pH change, and the ecological system develops towards an anaerobic environment instead of a good aerobic and anaerobic coexistence, resulting in a decrease in plant root activity and even necrosis. The microalgae cells in the microalgae biological soil remediation agent are basically single cells or single cell loosely combined cell groups, which can penetrate into the soil, adjust the activity of soil microorganisms, accelerate the decomposition of soil blocks, and effectively alleviate the phenomenon of too tight soil aggregate structure. In the deep soil with weak light, even if the microalgae cannot perform photosynthetic autotrophy due to insufficient light and the carried nutrients are exhausted and gradually die, they can also provide energy for other heterotrophic metabolizing microorganisms to continue to contribute to the symbiotic system of plant roots.

[0022] The purpose of the present application is also to provide a soil remediation method based on the aforementioned biological soil remediation agent, which is specifically as follows:

[0023] The microalgae biological soil remediation agent and the nutrient solution are mixed uniformly at a mass ratio of 8-10:80.

[0024] The nutrient solution comprises water and culture components accounting for 0.1-0.18% by weight; the culture components comprise sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, citric acid, ferric ammonium 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, ferric ammonium 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).

[0025] Further, the trace elements comprise 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).

[0026] When the culture components account for 0% by weight in the nutrient solution, the method comprises the steps of: dispersing and diluting the microalgae biological soil remediation agent; and soil drip irrigation is performed on the obtained dispersion;

[0027] When the culture components account for more than 0% by weight in the nutrient solution, the method comprises the steps of: mixing the microalgae biological soil remediation agent with the nutrient solution, and covering the corresponding containing container after uniform mixing; further, the covered containing container has a ventilation gap; further, the containing container can be a white container with a certain degree of transparency, such as a translucent white container. After being placed in the corresponding containing container for fermentation for 2-3 days, the fermentation temperature is 25-35℃, and the containing container is opened at least once a day for stirring during the fermentation process, and then the containing container is covered again for closed fermentation. After the fermentation is completed, the corresponding containing container is opened, and the microalgae expanded culture solution is obtained; the microalgae expanded culture solution is applied to the soil for soil remediation.

[0028] The biological soil remediation agent provided by the application is used for expanding the microalgae biological soil remediation agent, and a large amount of applicable microbial fertilizer can be obtained based on a small amount of microbial seed solution. The photosynthetic microbial liquid fertilizer is convenient for industrial application and reduces cost consumption. After the application of the biological soil remediation agent, the soil is inoculated with high-activity probiotics and original soil microalgae, the diversity of the soil microbial community is increased, the soil aggregate structure is improved, and the soil physical and chemical environment is improved. Compared with the prior art, the application has the following advantages:

[0029] 1) The application provides a sustainable benign underground microecosystem and a microalgae biological soil remediation agent to solve the problem that the microalgae biological activity is poor and cannot be sustained in the prior art under harsh soil conditions. The microalgae biological soil remediation agent is based on a porous active carrier with positive charges. The porous active carrier is obtained by compounding silicon dioxide on montmorillonite, and the porous carrier is modified to have negative charge NH2 - groups in the interlayer. Finally, the porous active carrier is loaded with chitosan cations to become a porous active carrier with positive charges, which is loaded with a large amount of Chlorella pyrenoidosa, so that it can survive and reproduce in a low-pH or high-pH environment, and the Chlorella pyrenoidosa liquid carried by it has low environmental sensitivity. The montmorillonite is compounded with silicon dioxide to improve the light transmittance, so that sufficient light can be obtained in the surface soil to start the photosynthetic autotrophic mode. In deep soil, the carrier adsorbs and carries energy to meet the heterotrophic and mixotrophic growth of the microalgae, so that the microalgae can maintain higher activity and form a sustainable benign underground microecosystem. Furthermore, under the protection of the porous active carrier, the microalgae biological activity can be protected in the case of high summer surface temperature, and the survival rate of the microalgae biological activity under high temperature is significantly improved. Moreover, the pH can be adjusted to be close to neutral during the growth and reproduction of the microalgae, which can resist the process of soil acidification. The microalgae biological soil remediation agent can penetrate into the soil, overcome the problem of long-term activity under insufficient light, and significantly adjust the activity of deep soil microorganisms to accelerate the decomposition of soil blocks and effectively alleviate the phenomenon of too tight soil aggregate structure.

[0030] 2) The microalgae-based soil remediation agent provided by this invention contains high-purity Chlorella proteoglycans, which can be used directly as a nutrient fertilizer or as a seed culture for propagation. The product is easy to use, and the propagated Chlorella solution has high concentration, strong vitality, rapid reproduction, and is economical. It ensures a high success rate for propagation in farmland, is safe and stable to use, easy to operate, and has low cultivation costs. After metabolism, the proteoglycans produce high-protein nutrients and provide crops with abundant nitrogen, phosphorus, potassium, and other nutrients, reaching directly to the root zone (the soil within 1-2 mm of the root surface). Metabolites stimulate root zone biological activity, rapidly promoting root growth and branching, resulting in strong and vigorous root hairs, improved nutrient absorption and supply capacity, faster crop greening and better growth, increased yield, and better economic benefits. Furthermore, this remediation agent is environmentally friendly, with a microalgae propagation solution pH of around 8.5, adjusting acidic soils, especially beneficial for farmland with continuous cropping and no fallow periods. It increases soil microbial diversity, promotes the growth of beneficial colonies, and helps restore the balance of the soil micro-ecosystem. When the protein-nucleated Chlorella and its metabolites are applied to the soil, the algal cells can colonize and grow in the soil and at the roots of plants. During the reproduction process, oxygen is produced for the roots to breathe, increasing soil porosity and preventing soil compaction.

[0031] 3) The microalgae-based soil remediation agent and corresponding soil remediation method provided by this invention can increase the diversity of soil microbial communities, improve soil aggregate structure, and improve the soil physicochemical environment. It increases the oxygen content and organic matter in the soil, promotes the formation of dominant beneficial microbial communities, and solves problems such as soil compaction, acidification, decline in agricultural product quality, continuous cropping, and pests and diseases, thereby promoting healthy plant growth, increasing crop yield, restoring crop properties, and improving crop quality. It improves the soil to achieve effects such as root strengthening, seedling vigor, nitrogen fixation and quality improvement, and acid regulation and antibacterial properties. Furthermore, it can be used in various ways, including dilution and dispersion, and propagation; especially the propagation method, which can quickly and effectively propagate the microorganisms in the biological soil remediation agent, thereby obtaining a large amount of applicable microbial liquid fertilizer based on a small amount of initial microbial inoculum.

[0032] 4) The biological soil remediation agent provided by this invention has the function of degrading toxic and harmful substances. Through the reproduction and growth of the living microorganisms contained therein in the soil, it decomposes the organic matter in the soil into small molecules that are easily absorbed by plants, thereby increasing the supply of plant nutrients, promoting plant growth, improving the quality of agricultural products and the agricultural ecological environment. It has the characteristics of strong bacterial activity, high concentration, rapid propagation, and being easy to use and cost-effective. It can activate the soil, enhance photosynthesis, and improve the yield and quality of crops. Attached Figure Description

[0033] Figure 1 A comparison of soil conditions on day 140 after applying the microalgae-based soil remediation agent prepared in Example 3 to the control area fertilized with ordinary fertilizer.

[0034] Figure 2 Comparison of root growth of sweet potatoes 8 days after fertilization;

[0035] Figure 3 Comparison of root growth of sweet potatoes 36 days after fertilization;

[0036] Figure 4 Comparison chart of sweet potato harvest;

[0037] Figure 5 The growth changes of sweet potatoes after applying the microalgae-based soil remediation agent prepared in Example 3;

[0038] Figure 6 A comparison chart of rice growth. Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0040] Example 1

[0041] A bio-microalgae soil remediation agent comprises a porous active carrier and bio-microalgae loaded on the porous active carrier. The porous active carrier is obtained by composited silica onto montmorillonite. Specifically, the porous carrier is obtained by composited silica onto montmorillonite, and the process involves adding 50 parts of calcium-based montmorillonite to 30 parts of deionized water, adding an excess of 1 mol / L KCl solution, and stirring for 12 hours to allow the Ca... 2+ K + Displacement, wash with deionized water until Cl is free. - After centrifugation, 50 parts of ethanol were added and stirred evenly to fully disperse the potassium-based montmorillonite. The temperature was raised to 40°C, and 25 parts of octadecyltrimethylammonium chloride were added. The mixture was stirred thoroughly and reacted for 2 hours. The suspension was removed and centrifuged at 3500 rpm. It was washed three times with deionized water and centrifuged again. The solid mixture at the bottom was removed, dried and ground at 60°C to obtain modified potassium-based montmorillonite. Tetraethyl orthosilicate, ethanol and deionized water were mixed at 50°C in a molar ratio of 1:4:4. Dilute hydrochloric acid was added dropwise to adjust the pH to 5, and the mixture was stirred to form a transparent sol. Five parts of the transparent sol were taken and 40 parts of modified potassium-based montmorillonite were slowly added. The mixture was stirred for 3 hours, heated to 60°C, and allowed to stand for 12 hours. Then it was cooled to room temperature and allowed to stand for 24 hours. After centrifugation and filtration, gel microspheres were obtained. The microspheres were washed three times with water, dried at 60°C, and then heated to 420°C at a rate of 2°C / min and kept at that temperature for 3 hours to obtain a porous carrier.

[0042] Further, the preparation of the amine-modified porous carrier with negative charge NH2 - group, specifically: 50 parts of the porous carrier are ultrasonically dispersed in 100 parts of toluene at room temperature, then heated to 80°C, 20 parts of (3-methylaminopropyl) trimethoxysilane are slowly added under stirring, and the stirring is continued for 6 hours; after the reaction is completed, it is cooled to room temperature, centrifuged, and washed with a large amount of ethanol and water, and dried to obtain the amine-modified porous carrier with negative charge NH2 - group;

[0043] Further, the preparation method of the positively charged porous active carrier, specifically: 100 parts of chitosan solution with a concentration of 5% is prepared by using 0.05% citric acid aqueous solution at a temperature of 60°C, 50 parts of the amine-modified porous carrier is added into the chitosan solution, and stirred for 100 minutes, after the reaction is completed, 10 parts of glucose and 15 parts of sodium alginate are further added for stirring for 80 minutes, and finally filtered, dried, ground, and filtered at 60 mesh to obtain the positively charged porous active carrier,

[0044] Take the Chlorella pyrenoidosa seed liquid, and the Chlorella pyrenoidosa density is 6×10 6 CFU / g or above, inoculate the Chlorella pyrenoidosa into the BG11 culture solution, and the inoculation amount is 5% to 15%, the initial density is 6×10 6 CFU / g, and the Chlorella pyrenoidosa is cultured under the conditions of a temperature of 25°C, pH=6.5, light intensity of 2300 lux, and 24 hours of light per day, and the culture time is 5 days to obtain the Chlorella pyrenoidosa liquid, wherein the viable bacterial count of the Chlorella pyrenoidosa is ≥10 9 CFU / g;

[0045] Add 20 parts of the positively charged porous active carrier into 100 parts of the Chlorella pyrenoidosa liquid, mix uniformly to obtain a dispersion liquid, and after 2 hours of static state, the precipitated part is washed with deionized water, and freeze-dried to obtain the microalgae biological soil remediation agent.

[0046] Example 2

[0047] A biological microalgae soil remediation agent includes 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: the porous carrier obtained by compounding silicon dioxide on montmorillonite, specifically: 50 parts of calcium-based montmorillonite is added into 30 parts of deionized water, an excessive amount of 1 mol / L KCl solution is added, and the stirring is continued for 10 hours to replace Ca 2+ with K + , and the washing with deionized water is continued until there is no Cl -After centrifugation, 50 parts of ethanol is added to stir evenly, so that the potassium-based montmorillonite is fully dispersed, and the temperature is raised to 40℃, 25 parts of octadecyl trimethyl ammonium chloride is added, and stirred fully, and reacted for 2 hours. The suspension is taken out and centrifuged at a speed of 3500 rpm, washed with deionized water for 3 times, and then centrifuged again. The bottom solid mixture is taken out and dried at 60℃, and ground to obtain the modified potassium-based montmorillonite; tetraethyl orthosilicate, ethanol and deionized water are mixed at a molar ratio of 1:4:4 at 40℃, and the pH is adjusted to 4.5 by adding dilute hydrochloric acid dropwise. A transparent sol is formed by stirring and reacting. 5 parts of the transparent sol is taken, and 40 parts of the modified potassium-based montmorillonite is slowly added. Stirring is performed for 3 hours, the temperature is raised to 60℃, and then the temperature is kept for 12 hours. Then the temperature is lowered to room temperature and kept for 24 hours. After centrifugal filtration, the gel microspheres are obtained, which are washed with water for 3 times, dried at 60℃, and then heated to 480℃ at a rate of 2℃ / min, and kept for 3 hours to obtain the porous carrier.

[0048] Further, the preparation of the amine-based porous carrier with negative charge NH2 - group between layers is specifically as follows: 50 parts of the porous carrier is ultrasonically dispersed in 100 parts of toluene at room temperature, and then the temperature is raised to 90℃. 30 parts of (3-methylaminopropyl)trimethoxysilane is slowly added under stirring, and stirring is continued for 6 hours. After the reaction is completed, the temperature is cooled to room temperature, centrifuged, and washed with a large amount of ethanol and water. The dried product is the amine-based porous carrier with negative charge NH2 - group between layers;

[0049] Further, the preparation method of the positively charged porous active carrier is specifically as follows: 0.05% citric acid aqueous solution is used to prepare 100 parts of a chitosan solution with a concentration of 3% at a temperature of 60℃. 40 parts of the amine-based porous carrier is added to the chitosan solution, and stirring is performed for 80 minutes. After the reaction is completed, 15 parts of glucose and 18 parts of sodium alginate are added, and stirring is continued for 80 minutes. Finally, the positively charged porous active carrier is obtained by filtering, drying, grinding, and filtering at 60 mesh.

[0050] The Chlorella pyrenoidosa seed liquid is taken, and the density of the Chlorella pyrenoidosa is 6×10 6 cfu / g or higher. The Chlorella pyrenoidosa is inoculated into the BG11 culture solution at an inoculation amount of 5% to 15%, and the initial density is 6×10 6 cfu / mL. The Chlorella pyrenoidosa is cultured at a temperature of 25℃, pH=6.7, light intensity of 2000 lux, and light illumination for 24 hours per day. The culture time is 4 days, and the Chlorella pyrenoidosa liquid is obtained, in which the viable cell count of the Chlorella pyrenoidosa is ≥10 9 cfu / g.

[0051] 30 parts of the positive charged porous active carrier is added into 100 parts of the Chlorella pyrenoidosa solution, mixed uniformly to obtain a dispersion liquid, after 2 hours of static state, the precipitated part is washed with deionized water, and frozen and dried to obtain the microalgae bioremediation agent.

[0052] Example 3

[0053] 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 silica on montmorillonite, specifically: the porous carrier obtained by compounding silica on montmorillonite, specifically: 50 parts of calcium-based montmorillonite is added into 30 parts of deionized water, an excess of 1 mol / L KCl solution is added, and stirring is continued for 10 hours to replace Ca 2+ with K + , and the KCl is washed away with deionized water until no Cl - is left; after centrifugal separation, 50 parts of ethanol is added and stirred uniformly to fully disperse the potassium-based montmorillonite, and the temperature is raised to 40℃, 25 parts of octadecyl trimethyl ammonium chloride is added, and fully stirred for 2 hours; the suspension is taken out and centrifugally separated at a speed of 3500 rpm, washed with deionized water for 3 times, and centrifugally separated again; the bottom solid mixture is taken out and dried at 60℃, and ground to obtain modified potassium-based montmorillonite; tetraethyl orthosilicate, ethanol and deionized water are mixed at a molar ratio of 1:4:4 at 40℃, and the pH of the mixed solution is adjusted to 4.5 by dropwise adding dilute hydrochloric acid, and the solution is stirred and reacted to form a transparent sol; 5 parts of the transparent sol is taken out, slowly added into 40 parts of the modified potassium-based montmorillonite, and stirred for 3 hours; the temperature is raised to 60℃, and the mixture is left to stand for 12 hours, and then the temperature is lowered to room temperature and left to stand for 24 hours; the gel microspheres are obtained by centrifugal filtration, washed with water for 3 times, dried at 60℃, and then the temperature is raised to 450℃ at a rate of 2℃ / min, and the porous carrier is obtained by keeping the temperature for 3 hours.

[0054] Further, an amine-modified porous carrier with negative charge NH2 - groups between layers is prepared, specifically: 50 parts of the porous carrier is ultrasonically dispersed into 100 parts of toluene at room temperature, and then the temperature is raised to 90℃, and 20 parts of (3-methylaminopropyl)trimethoxysilane is slowly added under stirring, and the stirring is continued for 6 hours; after the reaction is completed, the temperature is cooled to room temperature, centrifugally separated, and washed with a large amount of ethanol and water, and dried to obtain the amine-modified porous carrier with negative charge NH2 - groups between layers;

[0055] Further, the preparation method of the positively charged porous active carrier is as follows: 150 parts of chitosan solution with a concentration of 5% is prepared by using 0.05% citric acid aqueous solution at a temperature of 60℃, 50 parts of amine-based porous carrier is added into the chitosan solution, and stirring reaction is carried out for 90 minutes; after the reaction is completed, 15 parts of glucose and 20 parts of sodium alginate are further added for stirring reaction for 60 minutes; finally, the positively charged porous active carrier is obtained by filtering, drying, grinding and filtering with a mesh size of 60.

[0056] The Chlorella pyrenoidosa seed liquid is taken, and the density of the Chlorella pyrenoidosa is 6×10 6 CFU / g, the Chlorella pyrenoidosa is inoculated into the BG11 culture solution, the inoculation amount is 5%-15%, the initial density is 6×10 6 CFU / g, and the Chlorella pyrenoidosa is cultured under the conditions of a temperature of 25℃, pH=7.0, light intensity of 2500 lux, and 24 hours of light per day, and the culture time is 4 days, so that the Chlorella pyrenoidosa liquid is obtained, wherein the viable cell count of the Chlorella pyrenoidosa is ≥10 9 CFU / g.

[0057] 30 parts of the positively charged porous active carrier is added into 100 parts of the Chlorella pyrenoidosa liquid to obtain a dispersion liquid, the dispersion liquid is left to stand for 2 hours, the precipitated part is washed with deionized water, and the freeze-dried product is obtained as the microalgae biological soil remediation agent.

[0058] The microalgae biological soil remediation agent and the nutrient solution agent are mixed uniformly at a mass ratio of 8:80; when the culture component in the nutrient solution agent accounts for 0% by weight, the microalgae biological soil remediation agent is dispersed and diluted; and the obtained dispersion liquid is used for soil drip irrigation.

[0059] Example 4

[0060] The microalgae bioremediation agent prepared in Example 3 is mixed with the nutrient solution agent at a mass ratio of 8:80. When the culture component in the nutrient solution agent is 0.1%, the culture component includes sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, citric acid, ferric ammonium citrate, disodium EDTA, sodium carbonate, and trace elements. The weight ratio of sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, citric acid, ferric ammonium 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). 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). The nutrient solution agent is prepared according to the above ratio.

[0061] The microalgae bioremediation agent is mixed with the nutrient solution agent. After uniform mixing, the corresponding loading container is covered. Further, the loading container has a ventilation gap. Further, the loading container can be a white container with a certain degree of transparency. After fermentation in the corresponding loading container for 3 days, the fermentation temperature is 28°C. During the fermentation process, the loading container is opened at least once a day for stirring, and then the loading container is closed for further fermentation. After the fermentation is completed, the corresponding loading container is opened to obtain the microalgae expansion liquid. The microalgae expansion liquid is applied to the soil for soil remediation.

[0062] In Comparative Example 1, the obtained Chlorella pyrenoidosa liquid is directly used as the microalgae bioremediation agent, and the other conditions are the same as in Example 3.

[0063] In Comparative Example 2, calcium-based montmorillonite is directly used to replace the positively charged porous active carrier in an equal amount, and the other conditions are the same as in Example 3.

[0064] In Comparative Example 3, an amine-based porous carrier with a negative NH2 - group in the interlayer is directly used to replace the positively charged porous active carrier in an equal amount, and the other conditions are the same as in Example 3.

[0065] In Comparative Example 4, the active microalgae nutrient repair liquid product provided in Example 8 of Chinese Patent Publication No. CN109401977B is directly used.

[0066] Comparative Example 5, the ordinary nitrogen and phosphorus fertilizer was used to fertilize the sweet potato control area 3 times;

[0067] Comparative Example 6, the rice special fertilizer was used to fertilize the seawater rice control area 3 times.

[0068] Test data and result analysis

[0069] 1. Storage activity

[0070] Microalgae activity cell survival rate test of the microalgae bioremediation agent, the microalgae remediation products prepared by Example 1-3 and Comparative Example 1-4 were stored at room temperature in the dark environment for 10 days, 1 month, 3 months, 6 months, 7 active microalgae remediation products were observed under a microscope, the survival rate of microalgae was calculated by the ratio of the number of active microalgae cells at the time of observation to the number of active microalgae cells at the initial time, and the detection results are shown in Table 1.

[0071] The microalgae remediation products prepared by Example 1-3 and Comparative Example 1-4 were stored at 50°C in the dark environment for 1 day, 10 days, 15 days, 7 active microalgae remediation products were observed under a microscope, and the detection results are shown in Table 1.

[0072] Table 1 Detection results of microalgae active cell survival rate at room temperature

[0073]

[0074] Table 2 Detection results of microalgae active cell survival rate at high temperature

[0075]

[0076] As can be seen from Table 1, at room temperature, the active microalgae nutrient remediation liquid prepared by Example 1-3 and Comparative Example 1-4, the survival rate of Chlorella pyrenoidosa can still be maintained at more than 92% after 6 months; it shows that Chlorella pyrenoidosa has good compatibility and symbiosis, so that the microalgae bioremediation product has a long shelf life; when the temperature is increased to 50°C, the microalgae bioremediation agent of Example 1-3 can still maintain a survival rate of about 40% after 15 days, while the microalgae of Comparative Example 1-4 can hardly survive. This is mainly because Chlorella pyrenoidosa is very sensitive to temperature, and when it is in high temperature, it will enter a dormant state, and if it is continuously exposed, the cells will quickly lose activity. However, the microalgae bioremediation agent of Example 1-3 has a double protection of montmorillonite and sodium alginate, so that Chlorella pyrenoidosa hides in the micropores and enters a dormant state, but with the long-term continuous high temperature, the outer Chlorella pyrenoidosa cell membrane is severely damaged and loses activity, but compared with Comparative Example 1-4, the survival rate has been significantly improved.

[0077] 2. Soil activity detection

[0078] Take the contaminated acidic soil, pH value is about 5.0, evenly spread into 20cm after the square test site 1-4, the control group test site is the normal cultivated soil collected; the microalgae bioremediation product prepared in each example / comparative example is added into one of the groups of soil, mixed uniformly, and the mass of the microbial preparation added in each part of the soil accounts for 1% of the mass of the soil;

[0079] 1st square test site: light 2000, pH value is about 5.0, temperature 30℃;

[0080] 2nd square test site: light 5000 (12 hours of light, 12 hours of light avoidance), pH value is about 5.0, temperature 30℃;

[0081] 3rd square test site: light 500 (12 hours of light, 12 hours of light avoidance), pH value is about 5.0, temperature 30℃;

[0082] 4th square test site: light 500 (12 hours of light, 12 hours of light avoidance), pH value is about 5.0, surface temperature 50℃, simulate summer high temperature, 8 hours of high temperature 50℃, 16 hours of low temperature 30℃;

[0083] Control group test site: light 2000 (12 hours of light, 12 hours of light avoidance), pH value is about 7.0, temperature 30℃;

[0084] After fertilization, immediately take 5 parts of 1g surface soil samples, 5 parts of 1g 5cm deep soil samples, and 5 parts of 1g 20cm deep soil samples;

[0085] After 48 hours of fertilization, take 5 parts of 1g surface soil samples, 5 parts of 1g middle layer soil samples, and 5 parts of 1g 20cm deep soil samples from each test site;

[0086] After 48 hours of fertilization, take 5 parts of 1g surface soil samples, 5 parts of 1g middle layer soil samples, and 5 parts of 1g 20cm deep soil samples from each test site;

[0087] Table 3: amount of living cells in surface soil samples

[0088]

[0089] As can be seen from Table 3, when the surface is sufficiently illuminated, Examples 1-3 can overcome the inhibition at low pH, can quickly reproduce and maintain sufficient activity. When the light is insufficient, it can quickly start the abnormal matrix, so that in the case of slow reproduction, it can maintain sufficient activity. When high temperature occurs, it can enter the dormant period and maintain activity. This is because the growth rate of Chlorella pyrenoidosa is fast, the environmental tolerance is strong, and under the condition of nitrogen enrichment, it can synthesize a large amount of nitrogen-containing compounds (such as chlorophyll and protein) in the cell. In Examples 1-3, not only is Chlorella pyrenoidosa protected, but also nutrients are provided, so that it can grow quickly under insufficient or sufficient light, maintain activity for a long time, maintain a good sustainable growth micro-ecosystem. At the same time, studies have shown that the protonation or deprotonation reaction of the edge site of montmorillonite and the proton exchange reaction of the basal site endow montmorillonite with the ability to buffer PH. Within 60 hours, the pH value of the soil showed an upward trend, and the pH value of the microalgae-montmorillonite composite reaction system was relatively stable, which also showed that the addition of modified montmorillonite buffered the change of pH in the process of microalgae reproduction, and eliminated the negative effect of pH reduction on the growth and reproduction of microalgae. And Comparative Example 1 and Comparative Example 4, because the microalgae has no protective layer, and there is no sustainable release of nutrients, so its activity will quickly die and inactivate under insufficient light. And Comparative Examples 2 and 3, because the nutrients provided are insufficient and the protective effect is relatively weak, the growth of the microalgae becomes slow, and part of the microalgae living environment is poor and quickly inactivated.

[0090] Table 4 Amount of living cells in soil samples at a depth of 5 cm

[0091]

[0092] Table 5 Amount of living cells in soil samples at a depth of 20 cm

[0093]

[0094] As can be seen from Tables 3-5, the survival rate of viable bacteria number of the microalgae bioremediation agent in Examples 1-3 is high, and under high temperature and low light conditions, the heterotrophic mode can be started, and the microalgae can slowly multiply and grow, while the viable bacteria number in Comparative Examples 1-4 can keep less active. This is because although Chlorella pyrenoidosa can tolerate acidic environment for a period of time, it will not multiply in acidic and high temperature environment, and will be continuously inactivated in acidic and high temperature environment for more than 10 hours; and the inactivated bacteria are prone to die during storage, and the phenomenon of continuously decreasing viable bacteria with time occurs. The microalgae in the surface soil grow and multiply faster than those in the middle layer and at a depth of 20 cm because of sufficient light, and after the microalgae bioremediation agent of Examples 1-3 is applied to the middle layer soil, the light intensity of the agent is higher than that of the agent without compounded silicon dioxide in Comparative Examples 2 and 3, mainly because the light transmittance is good, so that the light transmitted through the soil gap can be fully utilized. When the soil depth continuously increases, the light utilization rate will be lower and lower, so that the growth and multiplication rate is further reduced, which is basically unrelated to the light intensity.

[0095] 3. Heavy metal ion adsorption capacity of microalgae

[0096] Test of heavy metal degradation capacity of the microalgae bioremediation product in soil: Take the contaminated soil around the smelting stone waste residue pile, dry in the air, crush, pass through a 20-mesh sieve, and mix thoroughly, then place in an autoclave, sterilize at 110°C for 100 minutes, and cool. Divide the sterilized soil into 24 parts, and place in sterilized containers. Divide the 24 parts of sterilized soil into 6 groups, 4 parts in each group. Take the microalgae bioremediation products prepared in Examples 1-3 and Comparative Examples 1-4, and add to one group of soil, each corresponding to one example / comparative example, mix thoroughly, and the mass of the microbial agent added to each part of soil accounts for 0.5% of the mass of the soil. After three months, detect the removal rate of exchangeable state of heavy metals in the treated soil, and take the average value of 3 parts in each group, and the results are shown in Table 6.

[0097]

[0098] From Table 6, it can be seen that the removal rates of various heavy metals in the soil by Examples 1-3 are relatively high, and the microalgae in Comparative Examples 1-4 have good removal rates of heavy metals. This is because the heavy metal ion adsorption capacity of microalgae can be attributed to a large number of functional groups such as carboxyl, sulfonic acid, sulfide, phosphorus group and the like on the cell surface. The large adsorption of heavy metal ions on the surface of microalgae cells can also fix heavy metal ions on the cell surface, hinder their entry into the cell, and maintain the activity of microalgae cells to a certain extent. At the same time, microalgae and other photosynthetic microorganisms can preferentially produce polypeptides that bind heavy metal ions, which is very beneficial to the biosorption of heavy metal ions. The microalgae biological soil remediation agent in Examples 1-3 has a porous active carrier with a positive charge, and the interaction between the montmorillonite and the microalgae in the system provides more binding sites for heavy metals, which can promote more heavy metals to be loaded onto the surface of the system. Under sufficient light, Comparative Example 2 uses unmodified montmorillonite, and the opaque montmorillonite can hinder photosynthesis, which weakens the growth and reproduction ability of microalgae in the soil, resulting in a low heavy metal removal rate. Although Comparative Example 3 is compounded with silicon dioxide to improve light transmittance, its heavy metal removal rate is higher than that of Comparative Example 2, but the montmorillonite has a negative charge, which repels the negative charge of the microalgae and affects the growth and reproduction ability of the microalgae. The microalgae biological soil remediation agent in Examples 1-3 can take full advantage of photosynthesis to enter the autotrophic mode, and can also start the mixotrophic mode. In addition, the synergistic effect of montmorillonite and chitosan can significantly enhance the removal rate of heavy metals.

[0099] 4. Monitoring of crop growth status

[0100] The effects of the fertilizers of Example 3 and Comparative Example 5 on Zhan purple sweet potato No. 3 were determined by taking Yingdie Village of Ke Lu Town in Leizhou City as the test site and Zhan purple sweet potato No. 3 as the test object. The soil condition after the microalgae biological soil remediation agent prepared by Example 3 was applied to the demonstration group for 140 days is shown in Figure 1 ; the soil condition after the fertilizer of Comparative Example 5 was applied to the control group for 140 days is shown in Figure 1 ; and the effects of the fertilizers of Example 3 and Comparative Example 5 on Zhan purple sweet potato No. 3 are shown in Figure 1From the comparison of soil conditions, the soil condition of the demonstration group of Example 3 was significantly improved after fertilization, and the soil condition changed from the original state of being hard and compact to being loose and porous. This is because the microalgae bioremediation agent improves the soil structure mainly through the aggregation of clay minerals and the adhesion of colloids. Microorganisms aggregate fine soil particles into clusters through extracellular polysaccharides and adhesive proteins in colloidal particles. This aggregate structure increases the porosity of the soil, providing better aeration and drainage conditions for plant root growth. At the same time, the aggregate structure also enhances the soil's resistance to wind and water erosion, maintaining the stability of the soil, and significantly increasing the quality and yield of crops. The average yield per mu of the demonstration group fertilized with Example 3 was 2592.8 kg, which was 851.5 kg more than the control group fertilized with Comparative Example 5. The microalgae bioremediation agent of Example 3 was used to fertilize the demonstration area at 3 kg per mu, and was used continuously for three times with an interval of 7 days. The ordinary nitrogen and phosphorus fertilizer of Comparative Example 5 was used to fertilize the control area at 3 kg per mu, and was used continuously for three times with an interval of 7 days. Figure 2 is a comparison chart of sweet potato root systems on the 8th day after fertilization 3 times, from Figure 2 It can be seen that the root system of Zhanzisuo No. 3 in the demonstration group is developed, and each root system has 3-5 more fruits than the control group. Figure 3 is a comparison chart of sweet potato root systems on the 36th day, from Figure 3 It can be seen that the root system of Zhanzisuo No. 3 in the demonstration group is developed, and each root system has 3-5 more fruits than the control group. Figure 4 is a harvest chart of Zhanzisuo No. 3, which also shows that the fruits in the demonstration group are large and numerous. Figure 5 is a chart showing the changes of Zhanzisuo No. 3 during growth, which reflects that after using the microalgae bioremediation agent, the soil condition improves, the organic matter content increases, and the crop growth is good, significantly increasing the yield per mu.

[0101] In the Xie General Base of Gangmen, the demonstration group of Example 4 was used to fertilize the microalgae expanded liquid prepared by Example 4, and the yield per mu reached 4732.64 kg, which was 904.76 kg higher than the control group using Comparative Example 1 as the microalgae bioremediation agent, an increase of 23.64%. All data were measured using standardized measurement: 3 meters of ridge, 1.2 meters wide.

[0102] Plant "Silky Red Potato", the demonstration group using the microalgae expanded liquid prepared by Example 4 for fertilization had a yield of 2999.09 kg per mu, an increase of 224.35 kg, and three points were measured for yield with two ridges of 5 meters.

[0103] The effects of the different microalgae fertilizers of Example 1-3 and Comparative Examples 1-4 and 6 on soil fertility were determined in Suixi County, Zhanjiang, Guangdong, with paddy rice as the test object. The soil organic matter content of the plough layer (0-20 cm) was 8.56 g / kg, the water content was 10.7%, and the heavy metal content was: total Cr 62.7 mg / kg, total Cu 38.5 mg / kg, and total As 6.5 mg / kg. The paddy rice was planted in the test site using the local conventional method as the control group (a total of 3 fertilizations, i.e., base fertilizer and two topdressing fertilizations, and the base fertilizer was rice special fertilizer (15-21-9) 50 kg / mu);

[0104] The paddy rice in the test group was managed in the same way as the control group, except that the microalgae biological soil remediation products of Example 1-3, Comparative Example 1-4, and 6 were applied at 4 kg / mu at the time of each growth period of the paddy rice when fertilization was needed (corresponding to the 3 fertilization periods of the control group), and the fertilization was performed 3 times. The paddy rice fertilized with Example 3 and Comparative Example 6 was compared at 56 days of growth, as shown in Figure 6 It can be seen that the paddy rice fertilized with Example 3 grew fast and had more white roots than the conventional rice of Comparative Example 6. The organic matter content, water content, and heavy metal content of the plough layer (0-20 cm) of the test group and the control group were measured at the time of harvest. The results are shown in Table 7.

[0105] Table 7 Effects of different groups on soil fertility and heavy metal content

[0106]

[0107] As can be seen from Table 7, the microalgae used as soil improvers can not only remove heavy metals, but also significantly improve soil structure and increase soil fertility. Microalgae can improve the water retention and air permeability of soil, providing a better growing environment for plants. This not only helps to improve crop yield and quality, but also reduces environmental problems such as soil erosion and water and soil loss. Microalgae not only survive and multiply in the soil and improve the biological structure of the soil, but also act as natural biological stimulants to promote plant growth. The application of microalgae in soil not only provides a nitrogen source, but also increases the content of other elements in the soil, improving soil fertility. Biological soil crust can strongly affect the organic matter content in the 0-5 cm soil layer, and can make the organic matter content in this layer significantly higher than that in the area without crust coverage.

[0108] The above description is only to illustrate the technical solutions of the present application and not to limit them. Any equivalent modifications and changes made by those skilled in the art to the technical solutions of the present application, as long as they do not deviate from the overall concept of the present application, are still within the scope of the present application.

Claims

1. A microalgae-based soil remediation agent, characterized in that: The microalgae-based soil remediation agent comprises a porous active carrier and microalgae loaded on the porous active carrier. The porous active carrier is obtained by composite silica onto montmorillonite, and the porous carrier is modified to have negatively charged NH2 interlayers. - The amino-modified porous carrier of the group is ultimately loaded with chitosan cations to become a positively charged porous active carrier; the microalgae include Chlorella proteoglycans solution, and the density of Chlorella proteoglycans is 6 × 10⁻⁶. 6 CFU / g or higher; Porous active carriers with positive charges are added to Chlorella pyrenoidosa solution and mixed evenly to obtain a dispersion. After standing for 1 to 2 hours, the precipitate is rinsed with deionized water and freeze-dried to obtain microalgae bio-soil remediation agent.

2. The microalgae-based soil remediation agent according to claim 1, characterized in that, The porous carrier mentioned above is a porous carrier obtained by compositing silica onto montmorillonite. Specifically, calcium-based montmorillonite is added to deionized water, and an excess of 1 mol / L KCl solution is added. The mixture is stirred for 10–12 hours to allow the CaO to precipitate. 2+ K + Displacement, wash with deionized water until Cl is free. - After centrifugation, ethanol was added and stirred until homogeneous, fully dispersing the potassium montmorillonite. The mixture was heated to 40°C, and octadecyltrimethylammonium chloride was added and stirred thoroughly. The reaction was allowed to proceed for 1–2 hours. The suspension was then removed and centrifuged at 3500 rpm. The mixture was washed 2–3 times with deionized water and centrifuged again. The solid mixture at the bottom was removed, dried at 60°C, and ground to obtain the modified potassium montmorillonite. The mass ratio of octadecyltrimethylammonium chloride to potassium montmorillonite was 1:

2. Tetraethyl orthosilicate, ethanol, and deionized water were mixed at a molar ratio of 1:4:4 at 40–50°C. Dilute hydrochloric acid was added dropwise to adjust the pH to 4–5, and the mixture was stirred to form a transparent sol. Modified potassium montmorillonite was slowly added, and the mixture was stirred for 2–3 hours. The temperature was raised to 60°C, and the mixture was allowed to stand for 12 hours. Then, it was cooled to room temperature and allowed to stand for 24 hours. After centrifugation and filtration, gel microspheres were obtained. The microspheres were washed with water 2–3 times, dried at 60°C, and then heated to 400–500°C at a rate of 2°C / min and kept at that temperature for 3 hours to obtain a porous carrier. The mass ratio of the transparent sol to potassium montmorillonite was 1:

8.

3. The microalgae-based soil remediation agent according to claim 1, characterized in that, The interlayer contains negatively charged NH2. - The preparation method of the amino-modified porous support is as follows: The porous support is ultrasonically dispersed in toluene at room temperature in an amount 2-3 times its weight. The temperature is then raised to 80-90°C, and (3-methylaminopropyl)trimethoxysilane is slowly added while stirring. Stirring continues for 5-6 hours. After the reaction is complete, the mixture is cooled to room temperature, centrifuged, washed with copious amounts of ethanol and water, and dried to obtain NH2 with negatively charged interlayer. - Aminated porous support with a functional group; wherein the mass ratio of (3-methylaminopropyl)trimethoxysilane to the porous support is 2-3:

5.

4. The microalgae-based soil remediation agent according to claim 1, characterized in that, The method for preparing the positively charged porous active carrier specifically involves: preparing a 3-5% chitosan solution using a 0.05% citric acid aqueous solution at a temperature of 50-60°C; and then adding negatively charged NH2 molecules to the interlayer. - Amination-modified porous carriers with functional groups are added to a chitosan solution and stirred for 80–100 minutes. After the reaction is complete, glucose and sodium alginate are added and the reaction is continued for another 50–80 minutes. Finally, the mixture is filtered, dried, ground, and filtered through a 60-mesh sieve to obtain a positively charged porous active carrier. The chitosan, sodium alginate, glucose, and negatively charged NH2 in the interlayer are present in the carrier. - The mass ratio of the amino-modified porous carrier is 3-8:15-20:10-15:40-50.

5. The microalgae-based soil remediation agent according to claim 1, characterized in that, Chlorella proteoglycans were inoculated into BG11 culture medium at a rate of 5%–15% and an initial density of 6 × 10⁶. 6 The cells / mL were cultured at 20–25℃, pH 6.5–7.1, light intensity 2000–3000 lux, and 24-hour light per day for 3–5 days to obtain a Chlorella proteoglycans solution with a viable count ≥10⁻⁶ cells / mL. 9 CFU / g; the mass ratio of positively charged porous active carrier to Chlorella proteoglycans solution is 2-3:

10.

6. A soil remediation method based on the microalgae biological soil remediation agent according to claim 1, wherein the microalgae biological soil remediation agent and the nutrient solution are mixed evenly at a mass ratio of 8-10:80; When the culture component accounts for 0% by weight in the nutrient solution, the steps include: dispersing and diluting the microalgae bio-soil remediation agent; and applying the obtained dispersion to soil drip irrigation. When the culture component accounts for more than 0% by weight in the nutrient solution, the process includes the following steps: mixing the microalgae biological soil remediation agent with the nutrient solution, and covering the mixture with a corresponding container after it is evenly mixed; leaving a ventilation gap in the covered container; the container is a white container with a certain degree of transparency, and the mixture is placed in the corresponding container for fermentation for 2-3 days at a fermentation temperature of 25-35℃. During the fermentation process, the container is opened at least once a day for stirring, and then the container is covered again for sealed fermentation. After the fermentation is completed, the corresponding container is opened to obtain the microalgae expansion solution; the microalgae expansion solution is applied to the soil for soil remediation.

7. The soil remediation method using the microalgae biological soil remediation agent according to claim 6, characterized in that, The nutrient solution comprises water and a culture component comprising 0.1–0.18% by weight. The culture component comprises sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, citric acid, ferric ammonium citrate, disodium EDTA, sodium carbonate, and trace elements. The weight ratio of sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, citric acid, ferric ammonium 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 microalgae 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).

Images