Composite algae cell as well as preparation method and application thereof
By incubating algal cells with amino-modified nanoparticles and emulsifying them with aqueous tanninic acid, nanoparticle algal cells with lipid membrane layer are formed, and complex algal cells with strong antibacterial ability are obtained, which solves the problem of insufficient antibacterial ability of existing algal cells and achieves efficient fertilizer delivery and soil nutrient regulation.
Patent Information
- Application Number
- CN202411741167.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-29
- Publication Date
- 2025-04-04
AI Technical Summary
When used as fertilizer, existing algae cells have insufficient antibacterial ability and cannot meet people's needs for antibacterial ability.
By incubating algal cells with amino-modified nanoparticles, algal cells with nanoparticle layer are formed, and mixed with emulsification with aqueous tanninic acid to form nanoparticle algal cells with lipid membrane layer. Cultivating under light, complex algal cells with strong antibacterial ability are obtained.
The antibacterial ability of complex algae cells is significantly improved, and the mass transfer efficiency and utilization rate of fertilizers are improved through ultrasonic atomization delivery method, effectively eliminate harmful bacteria, and regulate soil nutrient balance.
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Figure CN119751129A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of organic fertilizers, and in particular relates to a composite algae cell and a preparation method and application thereof. Background Art
[0002] Microalgae can provide nutrition, improve soil structure, promote microbial activity and enhance crop resistance as organic fertilizer in agriculture. Green algae contains a large amount of protein, vitamins, minerals and organic acids, which are essential nutrients for plant growth. After the green algae decompose, these nutrients can be directly absorbed and utilized by the root system of crops. After the green algae die, its cell wall can increase the porosity of the soil, improve the aeration and permeability of the soil, and is conducive to the development of the root system. The decomposition of green algae can promote the activity of beneficial microorganisms in the soil and help form a healthy soil ecosystem. Green algae can produce a series of growth regulators during its growth process, which can enhance the disease resistance of crops and improve the resistance of crops. However, with the increasing demand for antibacterial ability of crops, conventional algae cells can no longer meet people's demand for their antibacterial ability when used as fertilizers. Summary of the invention
[0003] In view of the above problems existing in the prior art, the present invention provides a composite algae cell and a preparation method and application thereof, which can provide a composite algae cell with strong antibacterial ability and improve the effect of fertilizer.
[0004] To achieve the above object, the technical solution provided by the present invention is as follows:
[0005] In a first aspect, the present application provides a method for preparing composite algal cells, comprising the following steps:
[0006] S1: incubating the algae cell solution with the amino-modified nanoparticles for 7-9 days to obtain algae cells with a nanoparticle layer;
[0007] S2: mixing the tannic acid aqueous solution with the algae cells having a nanoparticle layer, stirring to obtain an emulsion, centrifuging the emulsion to obtain the nanoparticle algae cells having a lipid membrane layer; culturing the nanoparticle algae cells having a lipid membrane layer under light to obtain the composite algae cells.
[0008] Optionally, the amino-modified nanoparticles are amino-modified ferrosoferric oxide particles.
[0009] Optionally, the preparation method of the amino-modified ferroferric oxide particles is: dissolve FeCl3 in a mixture of water and ethanol, add sodium acetate and polyethyleneimine to the mixture, ultrasonically treat it at 40-45°C for 1-1.5h, filter, wash and dry to obtain positively charged ferroferric oxide; dissolve the positively charged ferroferric oxide in an ethanol aqueous solution, ultrasonically treat it under acidic conditions, then add a silanization reagent to react at 50-60°C, wash and dry to obtain amino-modified ferroferric oxide particles.
[0010] Optionally, the algae cell solution is obtained by the following method: centrifuging the algae liquid, removing the supernatant and adding it to the culture medium, washing it, and allowing the washed algae liquid to stand to obtain a stable culture medium, which is the algae cell solution.
[0011] Optionally, the positively charged ferrosoferric oxide is dried in a vacuum at 60-70°C.
[0012] Optionally, the algal cell concentration in the culture medium after standing is 1×10 6 —1×10 7 Pieces / ml.
[0013] In a second aspect, the present application further provides a composite algal cell obtained by the preparation method described in the first aspect.
[0014] In a third aspect, the present application further provides the use of the composite algae cells described in the second aspect in an organic fertilizer, wherein the organic fertilizer is delivered by ultrasonic atomization.
[0015] Compared with the prior art, the present application provides at least the following beneficial effects:
[0016] The present invention obtains stable algae cells with a nanoparticle layer by incubating positively charged amino-modified nanoparticles with algae cells; then, the tannic acid aqueous solution is mixed and emulsified with the algae cells with the nanoparticle layer to obtain nanoparticle algae cells with a lipid membrane layer; after culturing under light, composite algae cells with oxygen in the core can be obtained through photosynthesis. The antibacterial ability of the composite algae cells obtained by the method of the present invention is greatly improved; by using the ultrasonic atomization delivery method for fertilization, the mass transfer efficiency of the microalgae fertilizer to the leaf surface is improved, and the fertilizer utilization rate is significantly improved.
[0017] The present invention not only helps to efficiently deliver oxygen, algae fertilizer and agricultural functional materials to the leaves of crops, but also can effectively eliminate harmful bacteria, thereby regulating the nutrient balance of farmland soil, providing a new method for solving the challenges of carbon fixation and emission reduction in current agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 Schematic diagram of the modification pathway of composite algae cells.
[0019] Figure 2 To fluorescently stain lipid membranes and proteins of the composite algae cells prepared in the example, confocal microscopy was used for imaging; (AB) is a scanning electron microscope (SEM) image of microalgae cells; (CD) is a projection electron microscope (TEM) image; (E) is chlorophyll a of microalgae cells showing red fluorescence under 543nm excitation; (F) is CDs / BOD labeled with FITC dye showing green fluorescence under 488nm excitation (BOD, bilirubin oxidase; CDs, carbon dots); (G) is Figure 2 Confocal fluorescence images merged from E and F; (H) Figure 2 Middle E and F are magnified images after merging.
[0020] Figure 3 This is the electron paramagnetic method (EPR) detection result of the composite algae cells prepared in this example.
[0021] Figure 4 This is the experimental flow chart of ultrasonic atomization delivery of algae cell-based microbubble organic fertilizer in this embodiment.
[0022] Figure 5 The results of the effects of different fertilizers on the growth of pakchoi are shown in Figure 2. Figure 5 aIn the laboratory; Figure 5 b is in the greenhouse. DETAILED DESCRIPTION
[0023] The present invention is further described in detail below in conjunction with the accompanying drawings:
[0024] The experimental methods used in the embodiments of the present invention are conventional methods unless otherwise specified.
[0025] The reagents and materials used in this example can all be purchased conventionally. The quantitative experiments involved in the examples were repeated at least three times, and the results were averaged.
[0026] Source of raw materials:
[0027] Chlorella algae liquid: from the Institute of Hydrobiology, Chinese Academy of Sciences.
[0028] Example
[0029] A method for preparing composite algae cells, comprising the following steps:
[0030] (1) Dissolve 40 mg of FeCl3·6H2O in 40 mL of a water / ethylene glycol mixture (volume ratio of 1:1), and then add 1.52 g of sodium acetate and 32 mg of polyethyleneimine (PEI) to the system. Then use an ultrasonic bath with an ultrasonic frequency of 40 kHz to ultrasonically treat the mixture to form a uniform solution. The temperature of the ultrasonic process is controlled at 40-45°C. After ultrasonic treatment for 1 hour, the product is collected by centrifugation and washed with distilled water and ethanol. The resulting product is then vacuum dried at 60°C for 24 hours to obtain positively charged nanoparticle magnetic ferroferric oxide;
[0031] 750 mg of positively charged nanoparticle magnetic ferroferric oxide was dispersed in 100 mL of 50% ethanol aqueous solution, the solution pH was adjusted to 4 with glacial acetic acid, and ultrasonic treatment was performed at room temperature for 10 minutes (40 kHz). Next, 1 mL of silanization reagent (3-aminopropyltrimethoxysilane in this embodiment) was added, reacted in a 60°C water bath under nitrogen protection, and mechanically stirred for 10 hours. After the reaction was completed, the product was washed with deionized water, anhydrous ethanol and anhydrous ether in sequence until there was no oily suspension. Finally, it was vacuum dried at 35°C to obtain amino-modified ferroferric oxide particles.
[0032] (2) Centrifuge the Chlorella algae solution (3000-4000rpm / min), then discard the supernatant to collect the algae cells, and add them to BG11 culture medium for washing once. Let the washed algae solution stand for 15-20 minutes to allow the algae cells to stabilize in the culture medium. This is the algae cell solution. Use an optical microscope to count the algae cells in the washed algae solution to determine that the algae cell concentration is generally 1×10 6 Pieces / mL;
[0033] The preparation method of BG11 culture medium is as follows: add 0.7g of BG11 powder into 1000ml of distilled water, heat it in a constant temperature water pot (80℃) for 2h, and then sterilize the heated culture medium.
[0034] (3) Take 50 ml of the algae cell solution obtained in step (2) and add 116 mg of the amino-modified ferrosoferric oxide particles obtained in step (1) and incubate them together for seven days to obtain green algae cells with a nanoparticle layer.
[0035] (4) Mix 9.5 mL of 2.5 mg / ml tannic acid aqueous solution with 50 mL of green algae cells with a nanoparticle layer. Use a high-speed homogenizer to emulsify the mixture and stir it at 10,000-12,000 rpm / min for 3-5 minutes (the speed in this embodiment is 11,000 rpm / min, 5 minutes) to obtain an emulsion. This process can promote the interaction between Fe³⁺ and phospholipids to form a metal-polyphenol composite network, thereby effectively fixing phospholipids on the cell surface. Subsequently, centrifuge at 3,600 rpm / min for 10 minutes to collect the nanomaterial algae cells with a lipid membrane layer. After culturing for 7 days under light conditions, the green algae will release oxygen through photosynthesis, and composite algae cells with oxygen in the core can be obtained.
[0036] The modification pathways of composite algal cells are as follows Figure 1 As shown in the figure, the multilayer structure consists of three parts from the inside to the outside: the core is the algae cell loaded with nanoparticle magnetic ferroferric oxide functional material, which is used to carry the functional material; the second layer is the protein layer, which is mainly responsible for carrying gas; the outermost layer is the envelope layer, which is used to stabilize the bubbles in the protein layer and play a role in protection and fixation. This design can ensure the controlled release of the functional material in the core and maintain the stability and functionality of the overall structure. The microscope imaging of the obtained composite algae cell is shown in Figure 2 It can be seen that the multilayer membrane structure oxygen-carrying algae microbubbles were successfully prepared.
[0037] The composite algae cells obtained in this example were tested by electron paramagnetic method (EPR), and the results were as follows: Figure 3 As shown in Figure 3a, the EPR spectrum characteristics of superoxide radicals (•O2⁻) at different time points (1 min, 5 min, 10 min, 20 min, 30 min). It can be seen that the typical four-line characteristic peaks appear in the figure, and the positions of these characteristic peaks are consistent with the standard EPR spectrum of superoxide radicals. Figure 1 This indicates that the capture agent DMPO reacts with •O2⁻ to generate a DMPO-•O2⁻ complex. At the same time, as the reaction time increases, the intensity of the characteristic peak gradually increases, indicating that the amount of superoxide radicals generated increases with time. Figure 3 Figure 3b shows the EPR spectra at multiple time points, showing a typical four-line equal peak feature (1:2:2:1 signal), which is a sign of DMPO-•OH complex formed by DMPO capturing hydroxyl radicals (•OH). The peak shape and splitting pattern of this signal are consistent with the characteristics of hydroxyl radicals. Therefore, the characteristics of Figure 3b indicate that the main free radical detected is hydroxyl radicals (•OH). Figure 3c shows a single, symmetrical EPR peak, which is significantly different from Figure 3 a and Figure 3The multi-line characteristics of free radicals in Figure b. The g value of the signal was calculated to be 2.002, which is consistent with the standard g value of oxygen vacancies, indicating that the signal may come from oxygen vacancies. The signal characteristics of Figure c are highly consistent with the performance of oxygen vacancies, indicating that Figure c represents the existence of oxygen vacancies. It can be seen that ROS are generated in the composite algae cells.
[0038] The algae cell-based microbubble organic fertilizer obtained in the example was delivered by ultrasonic atomization and compared with traditional fertilizers and sterilizers.
[0039] (1) Treatment of pakchoi with pesticides and fertilizers
[0040] Experimental group (DK group): Organic fertilizer and pesticide were delivered by ultrasonic atomization. The composite algae cells with oxygen in the core of Example 1 were used as organic fertilizer. Tannic acid and pesticide suspension were fully stirred at a ratio of 1:9 (tannic acid: pesticide) to obtain a mixed solution. The mixed solution was mixed with the composite algae cells to obtain a compound fertilizer with a composite algae cell content of 80 g / L. The compound fertilizer was then applied to the pakchoi at a spraying concentration of 250 ppm per square meter. Spraying frequency: spray once a week;
[0041] The experimental flow chart of delivering compound algae cell organic fertilizer by ultrasonic atomization is as follows Figure 4 As shown, the Labview control system is used to control the relevant parameters of the ultrasonic atomization delivery process to accurately regulate the process of ultrasonic atomization delivery of algae cell-based microbubble organic fertilizer. During the process, the Labview system controls the digital signal generator to generate a predetermined signal and transmits it to the power amplifier to provide sufficient power for ultrasonic atomization. The amplified signal is transmitted to the ultrasonic atomization equipment to atomize the algae cell-based microbubble organic fertilizer solution into fine particles, which are convenient for applying to plants or soil. The atomized microbubble organic fertilizer particles contact the vegetable samples for fertilization. In order to monitor the ultrasonic transmission effect, a hydrophone is used in the experiment to receive the ultrasonic signal and transmit the data to a digital oscilloscope. The digital oscilloscope displays the ultrasonic waveform in real time to analyze and verify the effect and accuracy of ultrasonic atomization;
[0042] Ultrasonic atomization parameters are controlled as follows:
[0043] Atomized particle size: 5-7 microns, 5 microns was used in the experiment
[0044] Spray distance: 20-50 cm, 30 cm was taken in the experiment.
[0045] Ultrasonic frequency range: 1.7 MHz to 2.4 MHz.
[0046] Control group A: using traditional fertilizer and pesticide delivery methods, using high-quality pesticides such as Sinochem nitrogen, phosphorus and potassium compound fertilizer (NPK20-20-20), spraying concentration: 5000ppm per square meter. The pesticide used is 75% chlorothalonil, and the spraying concentration per square meter is 250ppm. Spraying frequency: spray once a week;
[0047] The fertilizer delivery method uses an agricultural pressurized sprayer.
[0048] Control group B: using traditional fertilizer and pesticide delivery methods, the traditional pesticide is nitrogen-phosphorus-potassium compound fertilizer (NPK15-15-15) with a spraying concentration of 5000ppm per square meter, and the pesticide is 40% chlorothalonil with a spraying concentration of 250ppm per square meter, spraying once a week;
[0049] The fertilizer delivery method uses an agricultural pressurized sprayer.
[0050] Control Group C: Plants not sprayed with fertilizers or pesticides.
[0051] (2) Cultivation conditions after fertilization
[0052] After applying the fertilizer, the green vegetables were placed in a sunlight environment with a temperature of 22-24°C, a humidity of 65%-75%, and an air flow rate of about 1m / s. Their growth conditions and harmful bacteria on the leaves were regularly observed.
[0053] Plant growth 25 days after spraying Figure 5 It can be clearly seen that the growth of pakchoi grown with composite algae cells by ultrasonic atomization is better than that of the traditional culture group:
[0054] Plants in the CK group had the fastest growth rate, showed high growth vitality, thicker leaves, bright green and shiny leaves, and the best overall health, with almost no signs of wilting or disease. In contrast, although the growth rate of plants in the A group was slightly lower than that of the CK group, they still maintained good vitality, with slightly thinner but still healthy leaves, slightly lighter leaves, and good water and nutrient storage capacity.
[0055] In group B, the plant growth rate slowed down, the thickness of the leaves was significantly reduced, the leaf edges were slightly curled, showing certain signs of malnutrition, and the overall health was not as good as that of group A, and the vitality was reduced. The plants in group C showed the most obvious growth inhibition, the lowest growth rate, the thinnest leaves, and the color was yellow. The curling and wilting were more obvious, the overall health was poor, showing certain nutrient absorption disorders, and showing more diseases and insect pests.
[0056] This experiment can verify that the method of ultrasonic atomization delivery of composite algae cell-based organic fertilizer and pesticides shows a significant plant promotion effect. Compared with traditional fertilization and pesticide application methods, it has obvious advantages in plant growth. It is an efficient and environmentally friendly agricultural technology with broad application potential.
Claims
1. A method for preparing composite algal cells, characterized in that: The steps include: S1: incubating the algae cell solution with the amino-modified nanoparticles for 7-9 days to obtain algae cells with a nanoparticle layer; S2: mixing the tannic acid aqueous solution with the algae cells having a nanoparticle layer, stirring to obtain an emulsion, centrifuging the emulsion to obtain the nanoparticle algae cells having a lipid membrane layer; culturing the nanoparticle algae cells having a lipid membrane layer under light to obtain the composite algae cells.
2. The method for preparing composite algal cells according to claim 1, characterized in that: The amino-modified nanoparticles are amino-modified ferrosoferric oxide particles.
3. The method for preparing composite algal cells according to claim 2, characterized in that: The preparation method of the amino-modified ferroferric oxide particles is as follows: dissolving FeCl3 in a mixed solution of water and ethanol, adding sodium acetate and polyethyleneimine to the mixed solution, ultrasonically treating the mixed solution at 40-45°C for 1-1.5h, filtering, washing and drying to obtain positively charged ferroferric oxide; dissolving the positively charged ferroferric oxide in an ethanol aqueous solution, ultrasonically treating the mixed solution under acidic conditions, then adding a silanization agent to react at 50-60°C, washing and drying to obtain the amino-modified ferroferric oxide particles.
4. The method for preparing composite algal cells according to claim 1, characterized in that: The algae cell solution is obtained by the following method: centrifuging the algae liquid, removing the supernatant and adding it to the culture medium, washing it, and letting the washed algae liquid stand to obtain a stable culture medium, namely the algae cell solution.
5. The method for preparing composite algal cells according to claim 3, characterized in that: The positively charged ferroferric oxide is dried in a vacuum at 60-70°C.
6. The method for preparing composite algal cells according to claim 4, characterized in that: The algal cell concentration in the culture medium after standing was 1×10 6 —1×10 7 Pieces / ml.
7. A composite algal cell, characterized in that: The method is obtained by the preparation method described in any one of claims 1 to 6.
8. The composite algae cell according to claim 7 is used in an organic fertilizer, characterized in that: The organic fertilizer is delivered by ultrasonic atomization.