Nanopore vinasse carbon bioactive material loaded with microalgae and growth-promoting bacteria and preparation method of nanopore vinasse carbon bioactive material

Abstract

The invention discloses a microalgae and growth-promoting bacteria loaded nano-pore vinasse charcoal bioactive material and a preparation method thereof.The preparation method comprises the steps that wine making waste is subjected to high-temperature carbonization, vinasse charcoal with nano-pores is formed, and then the obtained vinasse charcoal is mixed with a nitric acid solution for acid modification; the physical and chemical properties of the modified vinasse biochar are fully utilized, microalgae and growth-promoting bacteria are carried, and the efficient combination of a nano material and microorganisms is realized by virtue of the biomineralization effect of the microorganisms, so that a micro-particle bioactive material with the particle size of 0.5-2mm and the water content of 30-40% is formed. The material not only realizes high-efficiency resource utilization of wine brewing wastes, but also has remarkable capabilities of reducing and fixing carbon and repairing desert sandy soil, can promote the growth of plant root systems, can effectively reduce pesticide residues on fruits and vegetables when being added into a microelement water-soluble fertilizer, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of preparation of bioactive materials, and in particular to a preparation method and application of a nanoporous distiller's grains charcoal bioactive material loaded with microalgae and growth-promoting bacteria. Background Art

[0002] With the enhancement of environmental awareness and the improvement of the demand for sustainable development, the resource utilization of brewing waste has become a research hotspot. In traditional technologies, brewing waste such as wine lees are usually treated by composting, feed processing, etc., but these methods often have problems of low treatment efficiency and low added value. In recent years, although some studies have attempted to carbonize wine lees at high temperatures and convert them into biochar with adsorption properties, how to further improve the application value of this biochar and give it more biologically active functions is still a technical problem that needs to be solved urgently. In particular, the loading efficiency of existing biochar materials on microalgae and growth-promoting bacteria and the functional characteristics of nanomaterials are unclear, which limits their wide application in ecological agriculture, environmental governance and other fields.

[0003] The main technical difficulty at present is that the initial pore structure of wine lees charcoal is poor, and it is difficult to meet the requirements of biological activated carbon for micropores and mesopore structures at the same time. Micropores are used to adsorb small molecules, while mesopores are conducive to microbial habitat and diffusion of large molecular organic matter. However, the existing pore distribution cannot take both into account, and the by-products produced in the carbonization process are also easy to block the pores, seriously affecting the adsorption performance and microbial loading capacity; and there are impurities such as incompletely carbonized organic components and metal ions on the surface of wine lees charcoal, which interfere with microbial attachment and chemical stability of activated carbon; at the same time, the types and number of surface functional groups do not meet the requirements of biological activated carbon, and suitable functional groups (such as carboxyl, hydroxyl, etc.) are crucial for microbial adsorption and fixation, and it is urgent to find a suitable modification method; different application scenarios have different requirements for microbial types and functions, and it is difficult to screen suitable microorganisms, and microorganisms need to adapt to the surface environment and living conditions of activated carbon; in addition, in actual use, biological activated carbon will affect the firmness of microbial fixation due to external forces, and it is urgent to explore efficient and stable immobilization technology.

[0004] In response to the above-mentioned difficulties, this patent has achieved key technological breakthroughs. With the help of advanced instruments and equipment and intelligent control systems, the carbonization temperature, time and other parameters are precisely controlled to ensure that the pore structure of biological activated carbon is developed and the performance is stable. Explore the use of high-efficiency modifiers to increase the number of surface functional groups and clean surface impurities to improve the adhesion performance of microorganisms. By accurately screening and controlling the types, inoculation time, proportion and environmental conditions of microalgae and growth-promoting bacteria, they produce sticky substances to enhance the binding force with the surface of activated carbon, while improving the adaptability to the chemical environment of the surface of activated carbon, ensuring an efficient and stable loading process. Based on these technological breakthroughs, a nanoporous wine lees charcoal bioactive material loaded with microalgae and growth-promoting bacteria has been successfully developed. This new bioactive material has the functions of reducing carbon and fixing carbon, repairing desert sand, promoting plant root growth, and reducing agricultural residues in fruits and vegetables. This series of technological innovations not only greatly enhances the application value of wine lees charcoal, but also provides strong material support for the sustainable development of ecological agriculture and environmental governance. Summary of the invention

[0005] Purpose of the invention: In order to solve the problems existing in the prior art, the present invention proposes a nanoporous distiller's grains charcoal bioactive material loaded with microalgae and growth-promoting bacteria and a preparation method thereof.

[0006] The preparation method of the present invention forms biochar with nanopores through high-temperature carbonization of winemaking waste, and cleverly utilizes the unique physicochemical properties of wine lees biochar to achieve efficient loading of microalgae and growth-promoting bacteria. With the help of the biomineralization of microorganisms, the nanomaterials and microorganisms are successfully combined to form a micro-particle bioactive material with a moderate particle size and a reasonable water content. This material not only effectively solves the problem of low efficiency in resource utilization of winemaking waste, but also provides new ideas for the development of ecological agriculture through its significant carbon reduction and carbon fixation, desert sand repair ability and the ability to promote plant root growth. At the same time, adding it to trace element water-soluble fertilizers can also effectively reduce agricultural residues in fruits and vegetables, showing broad application potential and market prospects.

[0007] Technical solution: To achieve the above purpose, the present invention adopts the following technical solution: a nanoporous distiller's grains charcoal bioactive material loaded with microalgae and growth-promoting bacteria, which is a modified distiller's grains biochar loaded with microalgae suspension and plant growth-promoting bacteria to form a micro-particle bioactive material with a particle size of 0.5-2mm and a water content of 30-40%.

[0008] The present invention also discloses a method for preparing the nanoporous distiller's grains charcoal bioactive material loaded with microalgae and growth-promoting bacteria, comprising the following steps:

[0009] (1) The microalgae suspension and the plant growth-promoting bacteria suspension are mixed in a volume ratio of 1.5:1-3:1, and then added to the modified vinasse biochar particles of 0.5-2 mm, and gently stirred to allow the microorganisms to be evenly adsorbed on the surface and pores of the modified vinasse biochar particles; the addition ratio of the modified vinasse biochar particles in the mixed suspension is 0.05-0.1 g / mL; the surface area of ​​the modified vinasse biochar particles is 25-35 m 2 / g, average adsorption pore size 2.5-3.5nm;

[0010] (2) The mixture is left to stand at a temperature of 20-30°C, a light intensity of 5000-10000 Lux, and a pH value of 7.0-8.0, allowing the microorganisms to naturally penetrate and attach to the modified wine lees biochar for 24-48 hours, and adjusting the moisture content of the final product to between 30-40%, thereby obtaining a nanoporous wine lees biochar bioactive material loaded with microalgae and growth-promoting bacteria.

[0011] Furthermore, the water content in step (2) is controlled by natural air drying or slight heating.

[0012] Furthermore, the preparation method of the modified distiller's grains biochar particles is to use distiller's grains as raw materials, perform high-temperature cracking under anaerobic conditions, and then perform nitric acid modification to obtain modified distiller's grains biochar particles with a porous structure and good adsorption properties.

[0013] Furthermore, the preparation method of the modified distiller's grains biochar particles specifically comprises the following steps:

[0014] 1) Preliminary screening of the lees to remove impurities and bulk substances;

[0015] 2) drying the screened lees at 103-107°C to constant weight;

[0016] 3) placing the dried wine lees in an oxygen-free environment at a pyrolysis temperature of 800° C. for high-temperature pyrolysis; after the pyrolysis is completed, naturally cooling to room temperature to obtain carbonized wine lees biochar;

[0017] 4) mixing the obtained vinasse biochar with a nitric acid solution to perform acid modification on the vinasse biochar;

[0018] 5) Grinding the acid-modified vinasse biochar into powder, sieving to obtain particles with a particle size of 0.5-2 mm, and using them as carriers for loading microalgae and growth-promoting bacteria.

[0019] Furthermore, the microalgae selected for the microalgae suspension in step (1) is one of Chlorella, Anabaena variabilis, and Nostoc, and the above microalgae have better growth efficiency and carbon dioxide absorption efficiency.

[0020] Furthermore, the specific method of acid modification in step 4) is: using 1-3 mol / L nitric acid solution, the mass ratio of wine lees biochar to nitric acid solution is 1:5-1:10, soaking and shaking, the oscillator is set to 100 times / min, and treating at 20-30°C for 6-10h.

[0021] Furthermore, the specific preparation method of the microalgae suspension in step (1) is as follows: select pure and unpolluted Chlorella, Anabaena variabilis, and diatom microalgae sources, use BG11 medium for Chlorella and Anabaena variabilis, use Zhu's No. 10 medium for diatoms, and culture the diatoms under the culture conditions of pH = 7-8, light intensity 8000 lux, light-dark ratio 12h:12h, temperature 20-30°C until the number of algae cells grows to 2×10 7 -5×10 7 / mL, the number of diatom cells grew to 2×10 5 -5×10 5 / mL; collect the algae by centrifugation or filtration, and resuspend the collected algae in a sterile buffer solution to adjust the concentration to 2×10 7 / mL can be put into use, thereby obtaining microalgae suspensions of Chlorella, Anabaena variabilis, and diatoms; the entire preparation process is carried out under sterile conditions.

[0022] Furthermore, the plant growth-promoting bacteria in the plant growth-promoting bacteria suspension in step (1) is selected as a mixture of Bacillus megaterium and Bacillus gelatinus in a mass ratio of 1:1-1.5:1; which has a better carbon fixation effect.

[0023] Furthermore, the specific preparation method of the plant growth-promoting bacterial suspension in step (1) is as follows: select pure and uncontaminated Bacillus megaterium and Bacillus jelly-like, use LB medium, culture under the conditions of pH = 7.0, temperature 30-37 ° C, and shaking speed 200r / min until the number of bacterial cells grows to 1×10 6 -5×10 6 / mL; collect the cells by centrifugation or filtration, remove the supernatant, and resuspend the cells with sterile saline or a specific buffer to adjust the concentration of the suspension to 1×10 6 / mL can be put into use. After sufficient stirring or oscillation, the Bacillus suspension is obtained. The aseptic operation specifications must be strictly followed during the preparation process to ensure the purity and quality of the suspension.

[0024] Furthermore, in step (1), the microalgae inoculum amount in the microalgae suspension in step (1) is 2×10 7 -5×10 7The inoculation amount of the plant growth-promoting bacteria in the plant growth-promoting bacteria suspension is 1×10 6 -5×10 6 cells.

[0025] The application of the above-mentioned nanoporous distiller's grains charcoal bioactive material loaded with microalgae and growth-promoting bacteria is specifically used for carbon reduction and fixation, repairing desert sand and promoting plant root growth, or adding it to trace element water-soluble fertilizer to reduce agricultural residues in fruits and vegetables.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0027] 1) High-temperature carbonization of brewing waste to form wine lees biochar with nanopores. In addition, further acid modification of wine lees biochar can increase the specific surface area of ​​wine lees biochar. Nitric acid can corrode the surface of biochar, making its originally dense structure loose and porous, thereby improving the adsorption capacity of biochar for heavy metal ions, organic pollutants, etc. Secondly, acid modification can adjust the surface functional groups of biochar. By reacting with nitric acid, more oxygen-containing functional groups such as carboxyl and hydroxyl groups can be introduced to the surface of biochar. These functional groups can change the surface charge properties of biochar, making it have better dispersibility and chemical reactivity in the solution, thereby enhancing the application effect of biochar in environmental remediation, catalysis and other fields. Acid modification helps to remove impurities in biochar. During the production process, biochar may have some ash or other impurity components. Nitric acid solution can dissolve and remove these impurities to a certain extent, further optimizing the performance of biochar.

[0028] 2) By making full use of the physicochemical properties of modified wine lees biochar, carrying microalgae and growth-promoting bacteria, and relying on the biomineralization effect of microorganisms, the efficient combination of nanomaterials and microorganisms is achieved to form tiny particles of biologically active materials. This not only realizes the efficient resource utilization of brewing waste, but also has significant carbon reduction and carbon fixation capabilities, and the ability to repair desert sand, and can promote the growth of plant roots. It has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a SEM photo of the modified distiller's grains biochar prepared in Example 1 of the present invention;

[0030] Figure 2 A comparison chart of carbon dioxide absorption rates in Examples 1-3 of the present invention and Comparative Examples 1-2;

[0031] Figure 3 is the adsorption curve of the modified vinasse biochar of the present invention;

[0032] Figure 4 This is a photo of the growth of tomatoes in the experimental group of Application Example 4 on the 20th day;

[0033] Figure 5 This is a photo of the growth of tomatoes in the control group of Application Example 4 on the 20th day. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with specific embodiments.

[0035] Embodiment 1:

[0036] The bioactive material of nanoporous vinasse charcoal loaded with microalgae and growth-promoting bacteria was prepared as follows:

[0037] (1) Preparation of modified wine lees biochar particles: Using wine lees as raw material, high-temperature pyrolysis is carried out under anaerobic conditions, and then acid modification is carried out to obtain wine lees biochar with a porous structure and good adsorption performance; the wine lees are preliminarily screened to remove impurities and large blocks; the screened wine lees are dried at 105°C to constant weight to reduce the moisture content for subsequent processing; the dried wine trough is placed in an anaerobic environment and pyrolysis is carried out at a high temperature of 800°C; after pyrolysis, it is naturally cooled to room temperature to obtain carbonized wine lees biochar to prevent structural damage of biochar due to excessive temperature; the obtained wine lees biochar is mixed with nitric acid solution and acid-modified; the specific method of acid modification is: using 2 mol / L nitric acid solution, the mass ratio of wine lees biochar to nitric acid solution is 1:8, soaking and shaking, the oscillator is set to 100 times / min, and the treatment is carried out at 25°C for 8h. The acid-modified lees biochar was ground into powder and sieved to obtain particles with a particle size of 0.5-2 mm as a carrier for loading microalgae and growth-promoting bacteria. After testing, the water content of the modified lees biochar was 12.33%, the ash content was 19.99%, the volatile matter was 22.98%, and the fixed carbon was 44.7%. The nanopore structure can be clearly observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For specific SEM photos, please refer to Figure 1 The BET specific surface area of ​​the modified nano-vinasse biochar is 30.4919 m 2 / g, the average adsorption pore diameter (4V / A) is 2.9557nm, and the average pore volume is 0.010180cm 3 / g. In addition, please refer to Figure 3 As shown in the figure, it is the carbon dioxide adsorption curve. The horizontal axis is the relative pressure and the vertical axis is the carbon dioxide adsorption amount. It is used to characterize the pore structure and adsorption performance of biochar. Figure 3 It can be seen that the modified distiller's grains biochar prepared by the present invention has good pore structure and adsorption properties.

[0038] (2) Preparation of microalgae suspension and plant growth-promoting bacteria suspension: The microalgae is one of Chlorella, Anabaena variabilis, and Nostoc, and the plant growth-promoting bacteria is a mixture of Bacillus megaterium and Bacillus gelatinus in a mass ratio of 1:1-1.5:1.

[0039] The specific preparation method of the microalgae suspension is as follows: pure and unpolluted Chlorella, Anabaena variabilis, and diatom microalgae sources are selected, Chlorella and Anabaena variabilis use BG11 culture medium, diatom uses Zhu's No. 10 culture medium, and diatom is cultured under the conditions of pH = 7-8, light intensity 8000 lux, light-dark ratio 12h:12h, temperature 20-30°C until the number of algae cells grows to 2×10 7 -5×10 7 / mL, the number of diatom cells grew to 2×10 5 -5×10 5 / mL; collect the algae by centrifugation or filtration, and resuspend the collected algae in a sterile buffer solution to adjust the concentration to 2×10 7 / mL can be put into use, thereby obtaining microalgae suspensions of Chlorella, Anabaena variabilis, and diatoms; the entire preparation process is carried out under sterile conditions to ensure the purity and quality of the microalgae suspension.

[0040] The specific preparation method of the plant growth-promoting bacterial suspension is as follows: select pure and uncontaminated Bacillus megaterium and Bacillus jelly-like. Use LB medium, culture for a period of time under the culture conditions of pH = 7.0, temperature 33 ° C, and shaker speed 200 r / min, and wait until the number of bacterial cells grows to 1×10 6 -5×10 6 The cells were collected by centrifugation, the supernatant was removed, and the cells were resuspended in sterile saline to adjust the concentration of the suspension to 1×10 6 / mL can be put into use. After sufficient stirring or oscillation, the Bacillus suspension is obtained. The aseptic operation specifications must be strictly followed during the preparation process to ensure the purity and quality of the suspension;

[0041] The microalgae inoculum in the microalgae suspension is 2×10 7 -5×10 7 The inoculation amount of the plant growth-promoting bacteria in the plant growth-promoting bacteria suspension is 1×10 6 -5×10 6 The microalgae suspension and the plant growth-promoting bacteria suspension are mixed at a volume ratio of 2:1, and then added to the 0.5-2 mm modified vinasse biochar particles, wherein the modified vinasse biochar particles are added to the mixed suspension at a ratio of 0.05-0.1 g / mL; gently stir to allow the microorganisms to be evenly adsorbed on the surface and pores of the biochar;

[0042] (3) The mixture was left to stand at 25°C, with a light intensity of 8000 Lux and a pH value of 7.5, to allow the microorganisms to naturally penetrate and attach to the biochar for 36 hours. The moisture content of the final product was adjusted to between 30% and 40% by natural air drying or slight heating to obtain a bioactive material of modified nano-vinasse biochar loaded with microalgae and plant growth-promoting bacteria.

[0043] Embodiment 2:

[0044] The other embodiments are the same as those in Example 1, except that the microalgae selected is Anabaena variabilis.

[0045] Embodiment 3:

[0046] The other embodiments are the same as those in Example 1, except that the microalgae selected are diatoms.

[0047] In order to select algae species that are suitable for modified nano-vinasse biochar and can give full play to their biological characteristics and efficient growth and carbon fixation, the following comparative experiments were conducted: according to the growth rate of microalgae, carbon dioxide absorption rate and adsorption rate of modified nano-vinasse biochar, suitable microalgae species were screened, and Chlorella used in Example 1, Anabaena variabilis used in Example 2, and Diatoms used in Example 3 were selected. At the same time, five different green algae or blue algae species were set as Scenedesmus in Comparative Example 1 and Synechococcus in Comparative Example 2 for comparative experiments to select the most suitable algae species. The culture conditions were pH = 7-8, light intensity 8000 lux, light-dark ratio 12h: 12h, temperature 25-30℃, and the growth conditions were recorded to judge the growth rate and carbon dioxide absorption rate to observe the carbon fixation capacity of different algae species.

[0048] Comparative Example 1:

[0049] Other embodiments are the same as those in Example 1, except that the microalgae selected is Scenedesmus.

[0050] Comparative Example 2:

[0051] The other embodiments are the same as those in Example 1, except that the microalgae selected is Synechococcus.

[0052] (I) Microalgae species selection experiment:

[0053] The growth records are shown in Tables 1 to 5:

[0054] Table 1 Chlorella growth record

[0055] Number of days 1 3 5 7 9 11 <![CDATA[OD 540 ]]> 0.529 1.785 2.507 3.413 3.682 3.977 DW / (g / L) 0.12 0.386 0.699 0.803 0.887 1.031 <![CDATA[Cell density / (10 7 pcs / mL)]]> 3.94 18.1 19.32 20.03 19.58 20.2

[0056] Table 2 Growth records of Anabaena variabilis

[0057] Number of days 1 3 5 7 9 11 <![CDATA[OD 560 ]]> 0.814 1.097 1.473 2.381 2.426 2.587 DW / (g / L) 15.4 15.9 16.74 17.79 18.18 19.04

[0058] Table 3 Diatom growth records

[0059]

[0060]

[0061] Table 4. Record of growth of Scenedesmus

[0062] Number of days 1 3 5 7 9 11 <![CDATA[OD 540 ]]> 0.507 1.287 1.349 1.435 1.518 1.704 <![CDATA[Cell density / (10 7 pcs / mL)]]> 1.4 3.77 4.32 4.28 4.19 3.97 DW / (g / L) 0.191 0.51 0.582 0.75 0.77 0.81

[0063] Table 5. Growth records of sphaerocephala

[0064] Number of days 1 3 5 7 9 11 <![CDATA[OD 540 ]]> 0.593 1.048 1.527 1.981 2.169 1.944 DW / (g / L) 0.32 0.624 0.936 1.293 1.477 1.42 <![CDATA[Cell density / (10 6 pcs / mL)]]> 8.79 10.05 18.72 20.66 21.87 21.45

[0065] In the table, DW is dry matter weight, Cell density is cell density. From the growth records of various algae species in Tables 1 to 5, it can be seen that the growth cycle of each algae species, among which Chlorella, diatoms, Scenedesmus, and Anabaena variabilis have higher growth rates.

[0066] In addition, the carbon dioxide absorption rate was measured by passing the same concentration of carbon dioxide gas into the sealed containers where the algae species were grown. After three days of aeration and cultivation, the gas in each sealed container was extracted and the carbon dioxide content was measured by gas chromatography, so as to obtain the carbon dioxide absorption rate of different algae species at the same time. Figure 2 As shown, it can be seen from high to low that they are: Chlorella, Anabaena, Nostoc, Synechocystis, and Scenedesmus.

[0067] Chlorella, Anabaena variabilis, and diatoms with good growth rate and carbon dioxide absorption rate were comprehensively selected for modified nano-vinasse biochar adsorption experiments, aiming to explore the growth and adsorption rate of microalgae on modified nano-vinasse biochar, so as to evaluate the potential of modified nano-vinasse biochar as a microalgae culture matrix. The specific experimental methods are as follows:

[0068] 10 g of the modified nano-vinasse biochar prepared in Example 1 was placed in a triangular flask, and a known concentration (2×10 7 -5×10 7 / mL) of microalgae culture solution, and record the initial microalgae concentration. Place the triangular flask in a constant temperature incubator, set at 25°C and 8000 lux for cultivation. Measure the absorbance of the microalgae solution every two days to determine the growth of the microalgae. At the same time, measure the mass change of the modified nano-vintners lees biochar from the beginning to the end to calculate the adsorption amount of the microalgae on the modified nano-vintners lees biochar. The results are shown in Tables 6-8.

[0069] Table 6 Adsorption effect of Chlorella on modified nano-vinasse biochar

[0070]

[0071] Table 7 Adsorption effect of diatoms on modified nano-vinasse biochar

[0072]

[0073] Table 8 Adsorption effect of Anabaena variabilis on modified nano-vinasse biochar

[0074]

[0075] The experimental results show that the three microalgae selected in Examples 1-3 of the present invention grow well on the modified nano-vinasse biochar and show a high adsorption rate, which further illustrates that the modified nano-vinasse biochar is a potential excellent microalgae culture matrix.

[0076] (II) Plant growth-promoting bacteria strain selection experiment:

[0077] In order to prove that the plant growth-promoting bacteria selected in Examples 1-3 of the present invention have efficient biological characteristics and efficient carbon fixation, the plant growth-promoting bacteria are evaluated according to the promotion of plant growth by plant growth-promoting bacteria, thereby further achieving carbon fixation through plant photosynthesis. A variety of bacteria that may have plant growth-promoting ability are collected from natural environments such as soil, plant roots, and humus, and are initially cultured. The collected strains were separated by the plate streaking method and purified on LB medium to obtain a single colony. Mixed culture was carried out with the screened microalgae species, and strains that can coexist with microalgae were preliminarily screened out: numbered 1-4; and the preliminarily screened strains were further tested for physiological characteristics, including the ability to produce indoleacetic acid (IAA), the ability to produce siderophores, the ability to solubilize phosphate, etc., as shown in Table 9. These characteristics can reflect the promoting effect of strains on plant growth. The screened strains were subjected to a compounding experiment to observe the effects of different strain combinations on tomato growth, as shown in Table 10. By comparing the effects of different combinations, the best strain combination is determined.

[0078] Table 9 Comparison of physiological characteristics of four strains

[0079] Strain No. IAA yield (μg / mL) Siderophore production (μg / mL) Phosphorus solubilizing capacity (mg / g) 1 20.54 0.85 26.30 2 21.07 0.69 20.14 3 13.26 0.32 12.47 4 14.69 0.38 10.46

[0080] The test results show that strains 1 and 2 have better physiological characteristics, and further strain combination experiments can be carried out to explore their promotion rate on plant growth.

[0081] Table 10 Experimental comparison of strain 1 and strain 2 used alone and in combination

[0082]

[0083] Where: Growth promotion rate = (growth of treatment group - growth of control group) / growth of control group × 100% (measure the growth promotion rates of root length, stem height and dry weight respectively and take the average value)

[0084] The results show that the best plant growth promotion rate can be obtained by selecting the combination of strains 1+2 with the highest plant growth promotion rate, and strains 1 and 2 are sequenced to obtain strain 1 as Bacillus megaterium and strain 2 as Bacillus gelatinus. That is, the strain combination used in Examples 1-3 of the present invention is the best. After subsequent experiments, the best effect can be obtained with a compound ratio of 1:1-1.5:1.

[0085] (III) Experiment on the selection of the mixing ratio of microalgae, plant growth-promoting bacteria and modified nano-vinasse biochar:

[0086] Different proportions of microalgae suspension, plant growth-promoting bacteria suspension and modified nano-vinasse biochar were set up, and the number of cells in each experimental group was 2×10 7 / mL of microalgae suspension, the cell number is 1×10 6 The plant growth-promoting bacteria suspension of 1000 g / mL was mixed at a volume ratio of 2:1, and the modified nano-vinasse biochar was added according to the ratio set in Table 11 and mixed evenly, and then inoculated into the culture medium respectively. Each experimental group was cultured at 30°C and 8000 lux for one week, and the growth of microalgae and plant growth-promoting bacteria was recorded. At the same time, the carbon dioxide fixation efficiency in each experimental group was determined to evaluate the effect of different mixing ratios on carbon reduction and carbon fixation.

[0087] Experimental group 1: the ratio of modified nano-vinasse biochar added to the microalgae and plant growth-promoting bacteria suspension was 0.1 g / mL;

[0088] Experimental group 2: the ratio of modified nano-vinasse biochar added to the microalgae and plant growth-promoting bacteria suspension was 0.05 g / mL;

[0089] Experimental group 3: the ratio of modified nano-vinasse biochar added to the microalgae and plant growth-promoting bacteria suspension was 0.01 g / mL;

[0090] Table 11 Comparison of carbon dioxide fixation rates at different mixing ratios

[0091]

[0092] The experimental results in Table 11 show that when the modified nano-vinasse biochar was added to the microalgae and plant growth-promoting bacteria suspension at a ratio of 0.1-0.05 g / mL, the cell density of the microalgae and the number of plant growth-promoting bacteria reached a high level, and the carbon dioxide capture efficiency was also the highest.

[0093] (IV) Acidification modification comparison experiment

[0094] I. Material preparation: The modified distiller's grains biochar particles prepared in step (1) of Example 1 were prepared using the same preparation method, except that the nitric acid was replaced by hydrochloric acid and sulfuric acid, respectively, to modify the performance of the distiller's grains biochar, to verify the mechanism of the influence of the unique chemical properties of nitric acid on the performance improvement of the modified biochar, and different acid concentrations and treatment time gradients were set to obtain a series of modified biochar particle samples.

[0095] II. Performance characterization methods: The specific surface area and pore structure of biochar were determined by specific surface area analysis (BET); the changes in functional groups on the surface of biochar were analyzed by Fourier transform infrared spectroscopy (FTIR); the surface morphology of biochar was observed by scanning electron microscopy (SEM); the content of carbon, nitrogen, oxygen and other elements in biochar was determined by elemental analyzer; the chemical state of elements on the surface of biochar was analyzed by X-ray photoelectron spectroscopy (XPS); and the adsorption capacity of biochar for specific pollutants (such as heavy metal ions or organic pollutants) was evaluated through adsorption experiments.

[0096] III. Experimental results:

[0097] Table 12 Comparison of specific surface area of ​​modified biochar particles prepared by acid treatment with different concentrations

[0098]

[0099]

[0100] 1) Specific surface area and pore structure: After analyzing the test data, the specific surface area of ​​the lees biochar modified by nitric acid is significantly increased, the average pore size is reduced, and the pore volume is increased, which is better than the biochar modified by hydrochloric acid and sulfuric acid. The specific surface area of ​​the biochar modified by nitric acid can reach 30m 2 / g, while those modified with hydrochloric acid and sulfuric acid are 23m 2 / g and 28m 2 / g. This is because nitric acid has strong oxidizing properties. During the acidification process, it can not only etch the surface of biochar to form more pores, but also undergo redox reactions with some carbonaceous components in biochar, causing the carbon skeleton structure to rearrange and promote the further development and refinement of pores. However, hydrochloric acid and sulfuric acid have relatively weak oxidizing properties, and their etching and structural rearrangement effects are not as obvious as nitric acid.

[0101] 2) Surface functional groups: FTIR spectra show that the content of oxygen-containing functional groups (such as carboxyl, hydroxyl, carbonyl, etc.) on the surface of nitric acid-modified biochar has increased significantly, and the types of functional groups are richer. Compared with hydrochloric acid and sulfuric acid-modified biochar, the functional group absorption peak intensity of the nitric acid-modified sample at a specific wave number is higher and the peak shape is sharper. The strong oxidizing property of nitric acid helps to introduce more active oxygen species on the surface of biochar and form more types of oxygen-containing functional groups. The increase of these functional groups is conducive to the chemical reaction and physical adsorption between biochar and pollutants, enhancing its adsorption performance.

[0102] 3) Elemental composition and chemical state: Elemental analysis and XPS results show that the nitrogen content of biochar modified with nitric acid is increased, and the nitrogen element exists in a variety of chemical states, including pyridinic nitrogen, pyrrolic nitrogen and graphitic nitrogen, among which the pyridinic nitrogen and pyrrolic nitrogen with higher chemical activity have relatively high contents. In contrast, hydrochloric acid and sulfuric acid modification have little effect on the nitrogen content and nitrogen chemical state of biochar. During the modification process, nitric acid can react with biochar through its nitrogen-containing groups, introduce nitrogen atoms into the biochar structure, and change its surface element chemical environment. This nitrogen doping effect enhances the surface activity and electronic conductivity of biochar, making it exhibit better performance in adsorption and catalysis.

[0103] 4) Adsorption performance: The adsorption test results show that the vinasse biochar modified by nitric acid acidification has a good adsorption performance on target heavy metal ions (such as Pb 2 +、Cd 2 +, etc.) or organic pollutants (such as methylene blue, tetracycline, etc.) are significantly higher than those of biochar modified with hydrochloric acid and sulfuric acid and original biochar. 2 +, the removal rate of nitric acid modified biochar can reach 93.82%, while that of hydrochloric acid and sulfuric acid modified biochar is 81.37% and 86.12% respectively, and that of original biochar is only 48.33%.

[0104] Combined with the above-mentioned modification effects in terms of specific surface area, functional groups and elemental composition, the larger specific surface area of ​​nitric acid-modified biochar provides more adsorption sites, and the rich oxygen-containing functional groups and nitrogen doping effects enhance its affinity for pollutants and chemical adsorption, thereby significantly improving its adsorption performance. However, the hydrochloric acid and sulfuric acid modified biochars have relatively weak modification effects in these aspects, and the improvement in adsorption performance is not as good as that of nitric acid modified biochar. The vinasse biochar modified by nitric acid has significant improvements in specific surface area, surface functional groups, elemental composition and adsorption performance compared with biochar modified by hydrochloric acid and other conventional acids. This improvement is mainly attributed to the unique strong oxidizing property of nitric acid, nitrogen doping ability and deep modification of the surface and structure of biochar. Therefore, the use of nitric acid to modify the performance of vinasse biochar makes the modified vinasse biochar have broader application prospects in agriculture, environmental remediation and other fields.

[0105] Application Example 5: Carbon Sequestration Efficiency

[0106] The nanoporous distiller's grains biochar loaded with bioactive materials (1 kg) prepared in Example 1 was placed in a closed reactor, and the reactor was cultured at 28°C and 8000 lux. The change in carbon dioxide concentration in the reactor was monitored every two days to determine the carbon dioxide absorption rate of the nanoporous distiller's grains biochar loaded with bioactive materials, as shown in Table 13.

[0107] Table 13 Carbon dioxide absorption rate of nanoporous lees biochar loaded with bioactive materials

[0108] Time (day) Carbon dioxide concentration in the reactor (ppm) Carbon dioxide absorption rate (%) 0 1500 0 2 924 38.4 4 782 47.8 6 522 65.2 8 495 67 10 455 69.6

[0109] It can be seen from Table 13 that the nanoporous distiller's grains biochar loaded with bioactive materials prepared by the present invention has good carbon fixation capacity.

[0110] Application Example 6: Promoting Plant Growth

[0111] The nanoporous distiller's grains biochar loaded with bioactive materials prepared in Example 1 was mixed with soil at a weight ratio of 1:3, and tomatoes were planted therein. Tomato seeds of the same high quality were selected and an experimental group and a control group (without the addition of nanoporous distiller's grains biochar loaded with bioactive materials) were set up at the same time. The growth of tomatoes, including plant height and leaf area, was regularly observed and recorded, as shown in Table 14.

[0112] Table 14 Growth of tomatoes grown with nanoporous distiller's grains biochar loaded with bioactive materials

[0113]

[0114] From the comparative data in Table 14, it can be seen that the experimental group with the addition of nanoporous lees biochar loaded with bioactive materials has obvious advantages in terms of plant height and leaf area. Please refer to Figure 4 (experimental group) and Figure 5 (Control group) is a comparison of the growth conditions of tomatoes on the 20th day. It can also be seen that the addition of the nanoporous distiller's grains biochar loaded with bioactive materials prepared in Example 1 of the present invention to the soil has a significant promoting effect on the growth of tomatoes.

[0115] Application Example 7: Reducing Pesticide Residues in Fruits and Vegetables

[0116] Cherry tomatoes and cucumbers were selected and grouped into three groups with similar growth conditions. Two replicates were set in each group. The three groups of fruits and vegetables were sprayed with the same concentration of pesticides to simulate the actual use of pesticides in production. The fruits and vegetables were placed outdoors in natural conditions and grown for one month.

[0117] 1) Watering treatment

[0118] A) Blank control group: Water was applied according to the conventional method, 500 mL each time, and watered once at regular intervals according to weather conditions to maintain appropriate soil moisture.

[0119] B) Control group 1: Use trace element water-soluble fertilizer for irrigation, 100 mL plus 500 mL of water each time, and the irrigation frequency is the same as the blank control group.

[0120] C) Experimental Group 2: The nanoporous distiller's grains biochar loaded with biologically active materials prepared in Example 1 of the present invention was mixed with trace element water-soluble fertilizer for irrigation. For every 100 mL of trace element water-soluble fertilizer, 500 mL of water and 10 g of nanoporous distiller's grains biochar loaded with biologically active materials were added. The irrigation frequency was the same as that of the control group and control group 1.

[0121] D) Pesticide residue detection

[0122] After 25 days, multiple fruit and vegetable samples were randomly selected from the three groups to test the pesticide organochlorine and organophosphorus residues in the fruits and vegetables. The test results of each sample were recorded, and the average pesticide residue content of each group was calculated.

[0123] Table 15 Effects of different treatments on pesticide residues in fruits and vegetables

[0124]

[0125] It can be seen from the experimental results in Table 15 that the nanoporous distiller's grains biochar loaded with bioactive materials prepared by the present invention mixed with trace element water-soluble fertilizer can effectively reduce agricultural residues in fruits and vegetables.

Claims

1. A nanoporous distiller's grains charcoal bioactive material loaded with microalgae and growth-promoting bacteria, characterized in that It is a tiny granular bioactive material with a particle size of 0.5-2mm and a water content of 30-40% formed by carrying microalgae suspension and plant growth-promoting bacteria to modify wine lees biochar.

2. A method for preparing the nanoporous vinasse charcoal bioactive material according to claim 1, comprising the following steps: The microalgae suspension and the plant growth-promoting bacteria suspension are mixed in a volume ratio of 1.5:1-3:1, and then added to the modified vinasse biochar particles of 0.5-2 mm, and gently stirred to allow the microorganisms to be evenly adsorbed on the surface and pores of the modified vinasse biochar particles; the addition ratio of the modified vinasse biochar particles in the mixed suspension is 0.05-0.1 g / mL; the surface area of ​​the modified vinasse biochar particles is 25-35 m 2 / g, average adsorption pore size 2.5-3.5nm; The mixture is left to stand at a temperature of 20-30°C, a light intensity of 5000-10000 Lux, and a pH value of 7.0-8.0, allowing the microorganisms to naturally penetrate and attach to the modified wine lees biochar for 24-48 hours, and the moisture content of the final product is adjusted to between 30-40% to obtain a bioactive material of nanoporous wine lees bioactive material loaded with microalgae and growth-promoting bacteria.

3. The method for preparing the nanoporous vinasse charcoal bioactive material according to claim 2, characterized in that The moisture content in step (2) is controlled by natural air drying or slight heating.

4. The method for preparing the nanoporous vinasse charcoal bioactive material according to claim 2, characterized in that: The preparation method of the modified vinasse biochar particles is to use vinasse as raw material, perform high-temperature cracking under anaerobic conditions, and then perform nitric acid modification to obtain modified vinasse biochar particles with a porous structure and good adsorption performance.

5. The method for preparing the nanoporous vinasse charcoal bioactive material according to claim 4, characterized in that The preparation method of the modified distiller's grains biochar particles specifically comprises the following steps: 1) Preliminary screening of the lees to remove impurities and bulk substances; 2) The screened lees are dried at 103-107°C to constant weight; 3) The dried wine lees are placed in an oxygen-free environment at a high temperature of 800°C for pyrolysis; after the pyrolysis is completed, the lees are naturally cooled to room temperature to obtain carbonized wine lees biochar; 4) mixing the obtained vinasse biochar with a nitric acid solution to perform acid modification on the vinasse biochar; 5) Grind the acid-modified wine lees biochar into powder, sieve to obtain particles with a particle size of 0.5-2 mm, and use them as carriers for loading microalgae and growth-promoting bacteria.

6. The method for preparing the nanoporous vinasse charcoal bioactive material according to claim 2, characterized in that: The microalgae selected for the microalgae suspension in step (1) is one of Chlorella vulgaris, Anabaena variabilis, and Nostoc sp.

7. The method for preparing the nanoporous vinasse charcoal bioactive material according to claim 6, characterized in that: The specific preparation method of the microalgae suspension in step (1) is as follows: select pure and unpolluted Chlorella, Anabaena variabilis, and diatom microalgae sources, use BG11 medium for Chlorella and Anabaena variabilis, use Zhu's No. 10 medium for diatoms, and culture the diatoms under the culture conditions of pH = 7-8, light intensity 8000 lux, light-dark ratio 12h:12h, temperature 20-30°C until the number of algae cells grows to 2×10 7 -5×10 7 / mL, the number of diatom cells grew to 2×10 5 -5×10 5 / mL; collect the algae by centrifugation or filtration, and resuspend the collected algae in a sterile buffer solution to adjust the concentration to 2×10 7 / mL can be put into use, thereby obtaining microalgae suspensions of Chlorella, Anabaena variabilis, and diatoms; the entire preparation process is carried out under sterile conditions.

8. The method for preparing the nanoporous vinasse charcoal bioactive material according to claim 2, characterized in that: The plant growth-promoting bacteria in the plant growth-promoting bacteria suspension in step (1) is a mixture of Bacillus megaterium and Bacillus gelatinus in a mass ratio of 1:1-1.5:

1.

9. The method for preparing the nanoporous distiller's grains charcoal bioactive material according to claim 8, characterized in that: The specific preparation method of the plant growth-promoting bacterial suspension in step (1) is as follows: select pure and uncontaminated Bacillus megaterium and Bacillus jelly-like, use LB medium, culture under the conditions of pH = 7.0, temperature 30-37 ° C, and shaking speed 200 r / min until the number of bacterial cells grows to 1×10 6 -5×10 6 / mL; collect the cells by centrifugation or filtration, remove the supernatant, and resuspend the cells with sterile saline or a specific buffer to adjust the concentration of the suspension to 1×10 6 / mL can be put into use, and after sufficient stirring or oscillation, the Bacillus suspension is obtained; the entire preparation process is carried out under sterile conditions.

10. The method for preparing nanoporous distiller's grains charcoal bioactive material according to claim 2, characterized in that: The microalgae inoculum in the microalgae suspension in step (1) is 2×10 7 -5×10 7 The inoculation amount of the plant growth-promoting bacteria in the plant growth-promoting bacteria suspension is 1×10 6 -5×10 6 cells.

Images