A nano-porous distiller's grains carbon bioactive material loaded with microalgae and growth-promoting bacteria and its preparation method

Abstract

The present invention discloses a nano-porous distiller's grains biochar biological active material loaded with microalgae and growth-promoting bacteria and a preparation method thereof. The brewing waste is subjected to high-temperature carbonization to form distiller's grains biochar with nano-pores, and then the obtained distiller's grains biochar is mixed with a nitric acid solution for acid modification. By making full use of the physical and chemical properties of the modified distiller's grains biochar, microalgae and growth-promoting bacteria are carried, and with the help of the biomineralization of microorganisms, the efficient combination of nano-materials and microorganisms is realized, forming a tiny particle biological active material with a particle size of 0.5-2 mm and a moisture content of 30-40%. This material not only realizes the efficient resource utilization of brewing waste, but also has remarkable carbon reduction and carbon sequestration and desert sandy soil restoration capabilities, can promote the growth of plant roots, and can effectively reduce the pesticide residues in fruits and vegetables when added to trace element water-soluble fertilizers, having broad application prospects.

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 nano-porous distiller's grains biochar bioactive material loaded with microalgae and growth-promoting bacteria. Background Art

[0002] With the enhancement of environmental protection 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 distiller's grains is usually treated by composting, feed processing, etc., but these methods often have problems such as low treatment efficiency and low added value. In recent years, although there have been studies attempting to subject distiller's grains to high-temperature carbonization treatment to convert them into biochar with adsorption properties, how to further improve the application value of this biochar and endow it with more bioactive functions remains a technical problem to be solved urgently. In particular, the loading efficiency of microalgae and growth-promoting bacteria in existing biochar materials and the functional characteristics of nanomaterials are not clear, which limits their wide application in the fields of ecological agriculture, environmental governance, etc.

[0003] Currently, the main technical problems are reflected in the poor initial pore structure of distiller's grains biochar, which is difficult to simultaneously meet the requirements of bioactive carbon for micropore and mesopore structures. Micropores are used to adsorb small molecules, while mesopores are conducive to the habitation of microorganisms and the diffusion of macromolecular organic matter. However, the existing pore distribution cannot take both into account, and the by-products generated in the carbonization process are also likely to block the pores, seriously affecting the adsorption performance and microbial loading capacity; moreover, there are impurities such as uncompletely carbonized organic components and metal ions on the surface of distiller's grains biochar, which interfere with the attachment of microorganisms and the chemical stability of activated carbon; at the same time, the types and amounts of surface functional groups also do not meet the requirements of bioactive carbon. Appropriate functional groups (such as carboxyl groups, hydroxyl groups, etc.) are crucial for the adsorption and fixation of microorganisms, and it is urgent to find a suitable modification method; different application scenarios have different requirements for the types and functions of microorganisms, and it is difficult to screen suitable microorganisms, and the microorganisms need to adapt to the surface environment and survival conditions of activated carbon; in addition, in actual use, the bioactive carbon will affect the firmness of microbial fixation due to external forces, and it is urgent to explore an efficient and stable immobilization technology.

[0004] In response to the above problems, this patent has achieved key technological breakthroughs. By means of advanced instrument equipment and intelligent control systems, parameters such as carbonization temperature and time are precisely controlled to ensure that the bioactive carbon has a well-developed pore structure and stable performance. The exploration and utilization of highly efficient modifiers are carried out to increase the number of surface functional groups and clean surface impurities, thereby enhancing the microbial adhesion performance. By precisely screening and controlling the types, inoculation time, ratio, and environmental conditions of microalgae and growth-promoting bacteria, sticky substances are produced to enhance the binding force with the activated carbon surface, and at the same time, the adaptability to the chemical environment of the activated carbon surface is improved, ensuring an efficient and stable loading process. Based on these technological breakthroughs, a nano-porous distillers' grains bioactive carbon material loaded with microalgae and growth-promoting bacteria has been successfully developed. This new bioactive material has functions such as carbon reduction and fixation, desert sandy soil restoration, promotion of plant root growth, and reduction of pesticide residues in fruits and vegetables. This series of technological innovations not only greatly improves the application value of distillers' grains biochar but also provides strong material support for the sustainable development of ecological agriculture and environmental governance. Summary of the Invention

[0005] Object of the Invention: In order to solve the problems existing in the prior art, the present invention provides a nano-porous distillers' grains bioactive carbon material loaded with microalgae and growth-promoting bacteria and its preparation method.

[0006] The preparation method of the present invention forms biochar with nano-pores by high-temperature carbonization of brewing waste, and by skillfully utilizing the unique physical and chemical properties of distillers' grains biochar, efficient loading of microalgae and growth-promoting bacteria is achieved. With the biomineralization of microorganisms, nano-materials and microorganisms are successfully combined tightly to form a small-particle bioactive material with a moderate particle size and reasonable moisture content. This material not only effectively solves the problem of low resource utilization efficiency of brewing waste but also provides new ideas for the development of ecological agriculture through its remarkable carbon reduction and fixation, desert sandy soil restoration capabilities, and the characteristics of promoting plant root growth. At the same time, when added to trace element water-soluble fertilizers, it can effectively reduce pesticide residues in fruits and vegetables, showing broad application potential and market prospects.

[0007] Technical Solution: To achieve the above object, the present invention adopts the following technical solution: A nano-porous distillers' grains bioactive carbon material loaded with microalgae and growth-promoting bacteria is a small-particle bioactive material with a particle size of 0.5 - 2 mm and a moisture content of 30 - 40% formed by modified distillers' grains biochar carrying microalgae suspension and plant growth-promoting bacteria.

[0008] The present invention also discloses a preparation method of the above nano-porous distillers' grains bioactive carbon material loaded with microalgae and growth-promoting bacteria, which includes the following steps:

[0009] (1) Mix the microalgae suspension and the plant growth-promoting bacteria suspension at a volume ratio of 1.5:1 - 3:1, and then add them to the modified distiller's grains biochar particles with a size of 0.5 - 2 mm. Gently stir to evenly adsorb the microorganisms on the surface and in the pores of the modified distiller's grains biochar; the addition ratio of the modified distiller's grains biochar particles in the mixed suspension is 0.05 - 0.1 g / mL; the surface area of the modified distiller's grains biochar particles is 25 - 35 m 2 / g, and the average adsorption pore diameter is 2.5 - 3.5 nm;

[0010] (2) Under static conditions, set the temperature at 20 - 30 °C, the light intensity at 5000 - 10000 Lux, and maintain the pH value at 7.0 - 8.0. Let the microorganisms naturally penetrate and attach to the inside of the modified distiller's grains biochar for 24 - 48 h, and adjust the moisture content of the final product to be between 30 - 40%, to obtain the bioactive material of the nano-porous distiller's grains charcoal bioactive material loaded with microalgae and growth-promoting bacteria.

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

[0012] Furthermore, the preparation method of the modified distiller's grains biochar particles is using distiller's grains as the raw material, performing high-temperature pyrolysis under anaerobic conditions, and then performing nitric acid modification to obtain the modified distiller's grains biochar particles with a porous structure and good adsorption performance.

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

[0014] 1) Preliminarily screen the distiller's grains to remove impurities and large pieces;

[0015] 2) Dry the screened distiller's grains at 103 - 107 °C until constant weight;

[0016] 3) Under an anaerobic environment, pyrolyze the dried distiller's grains at a pyrolysis temperature of 800 °C; after pyrolysis is completed, naturally cool to room temperature to obtain the carbonized distiller's grains biochar;

[0017] 4) Mix the obtained distiller's grains biochar with nitric acid solution to perform acid modification on the distiller's grains biochar;

[0018] 5) Grind the acid-modified distiller's grains biochar into powder, and screen to obtain particles with a particle size of 0.5 - 2 mm as the carrier for loading microalgae and growth-promoting bacteria.

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

[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 7cells, and the inoculation amount of the plant growth-promoting bacteria in the plant growth-promoting bacteria suspension is 1×10 6 -5×10 6 cells per mL.

[0025] The application of the above-mentioned nano-porous distillers grains biochar bioactive material loaded with microalgae and growth-promoting bacteria is specifically used for carbon reduction and carbon sequestration, repairing desert sandy soil, promoting plant root growth, or adding it to trace element water-soluble fertilizers to reduce pesticide residues in fruits and vegetables.

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

[0027] 1) The brewing waste is subjected to high-temperature carbonization to form distillers grains biochar with nano-pores. In addition, further acid modification of the distillers grains biochar can increase the specific surface area of the distillers grains biochar. Nitric acid can corrode the surface of the biochar, making its originally relatively dense structure become loose and porous, thereby improving the adsorption capacity of the biochar for heavy metal ions, organic pollutants, etc. Secondly, acid modification can adjust the surface functional groups of the biochar. By reacting with nitric acid, more oxygen-containing functional groups such as carboxyl groups and hydroxyl groups can be introduced onto the biochar surface. These functional groups can change the surface charge properties of the biochar, making it have better dispersibility and chemical reaction activity in solution, thereby enhancing the application effect of the biochar in the fields of environmental remediation, catalysis, etc. Acid modification helps to remove impurities in the biochar. During the production process, the biochar may retain some ash or other impurity components, and the nitric acid solution can play a role in dissolving and removing these impurities, further optimizing the performance of the biochar.

[0028] 2) By making full use of the physical and chemical properties of the modified distillers grains biochar, microalgae and growth-promoting bacteria are loaded, and with the help of the biomineralization of microorganisms, the efficient combination of nano-materials and microorganisms is realized, forming a micro-particle bioactive material. It not only realizes the efficient resource utilization of brewing waste, but also has significant carbon reduction and carbon sequestration capabilities, the ability to repair desert sandy soil, and can promote plant root growth, with broad application prospects. Description of the Drawings

[0029] Figure 1 It is the SEM photograph of the modified distillers grains biochar prepared in Example 1 of the present invention;

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

[0031] Figure 3 It is the adsorption curve of the modified distillers grains biochar of the present invention;

[0032] Figure 4 It is the photograph of the growth situation of the experimental group tomatoes on the 20th day in Application Example 4;

[0033] Figure 5 It is a photo of the growth of tomatoes in the control group on the 20th day in Application Example 4. Detailed implementation manners

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

[0035] Embodiment 1:

[0036] Prepare a bioactive material of a nano-porous distiller's grains biochar loaded with microalgae and growth-promoting bacteria according to the following steps:

[0037] (1) Preparation of modified distiller's grains biochar particles: Using distiller's grains as raw materials, pyrolyze them under anaerobic conditions at high temperature, and then perform acid modification to obtain distiller's grains biochar with a porous structure and good adsorption performance; preliminarily screen the distiller's grains to remove impurities and large substances; dry the screened distiller's grains at 105 °C until constant weight; to reduce the moisture content for subsequent processing; pyrolyze the dried distiller's grains in an anaerobic environment at a pyrolysis temperature of 800 °C; after pyrolysis is completed, naturally cool to room temperature to obtain carbonized distiller's grains biochar; to prevent the structure of the biochar from being damaged due to excessive temperature; mix the obtained distiller's grains biochar with nitric acid solution to perform acid modification on the distiller's grains biochar; the specific method of acid modification is: use a 2 mol / L nitric acid solution, the mass ratio of distiller's grains biochar to nitric acid solution is 1:8, soak and shake, set the oscillator to 100 times / min, and process at 25 °C for 8 h. Grind the acid-modified distiller's grains biochar into powder, and sieve to obtain particles with a particle size of 0.5 - 2 mm as the carrier for loading microalgae and growth-promoting bacteria. After testing, the obtained modified distiller's grains biochar has a water content of 12.33%, an ash content of 19.99%, a volatile content of 22.98%, and a fixed carbon content of 44.7%. Through characterization means such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the nano-porous structure can be clearly observed. For the specific SEM photos, please refer to Figure 1 as shown. The BET specific surface area of the modified nano-distiller's grains biochar is 30.4919 m 2 / g, the adsorption average pore diameter (4V / A) is 2.9557 nm, and the average pore volume is 0.010180 cm 3 / g. In addition, please refer to Figure 3 as shown, which is the carbon dioxide adsorption curve. The abscissa is the relative pressure, and the ordinate is the carbon dioxide adsorption amount, used to characterize the pore structure and adsorption performance of the biochar. It can be seen from Figure 3 that the modified distiller's grains biochar prepared by the present invention has good pore structure and adsorption performance.

[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) Under static conditions, set the temperature of the mixture to 25 °C, the light intensity to 8000 Lux, and maintain the pH value at 7.5. Allow the microorganisms to naturally penetrate and adhere to the interior of the biochar for 36 h. Adjust the moisture content of the final product to be between 30% and 40% by natural air drying or slight heating to obtain a bioactive material of modified nano distiller's grains biochar loaded with microalgae and plant growth-promoting bacteria.

[0043] Example 2:

[0044] Other implementation methods are the same as those in Example 1, except that the microalgae selected are Anabaena variabilis.

[0045] Example 3:

[0046] Other implementation methods are the same as those in Example 1, except that the microalgae selected are diatoms.

[0047] In order to select microalgae species that are adapted to the modified nano distiller's grains biochar and can fully exert its biological characteristics and efficient growth and carbon fixation, the following comparative experiments were carried out: Screen suitable microalgae species according to the growth rate of microalgae, carbon dioxide absorption rate, and adsorption rate of the modified nano distiller's grains biochar. Select Chlorella vulgaris used in Example 1, Anabaena variabilis used in Example 2, and diatoms used in Example 3. At the same time, set Control Example 1 as Scenedesmus obliquus and Control Example 2 as Pediastrum boryanum. Five different green algae or cyanobacteria species were used for comparative experiments to select the most suitable microalgae species. The culture conditions were pH = 7 - 8, light intensity 8000 lux, light-dark ratio 12 h:12 h, and temperature 25 - 30 °C. Record the growth situation to judge the growth rate and carbon dioxide absorption rate to observe the carbon fixation ability of different microalgae species.

[0048] Control Example 1:

[0049] Other implementation methods are the same as those in Example 1, except that the microalgae selected are Scenedesmus obliquus.

[0050] Control Example 2:

[0051] Other implementation methods are the same as those in Example 1, except that the microalgae selected are Pediastrum boryanum.

[0052] (I) Microalgae species selection experiment:

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

[0054] Table 1 Growth record of Chlorella vulgaris

[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 cells / mL)]]> 3.94 18.1 19.32 20.03 19.58 20.2

[0056] Table 2 Growth record 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] Record of the growth of diatoms in Table 3

[0059]

[0060]

[0061] Record of the growth of Scenedesmus in Table 4

[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 cells / 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] Record of the growth of Synechococcus in Table 5

[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 cells / mL)]]> 8.79 10.05 18.72 20.66 21.87 21.45

[0065] In the table, DW is the dry matter weight, and Cell density is the cell density. From the growth records of each algal species in Tables 1 - 5, the growth cycles of each algal species can be seen. Among them, the growth rates of Chlorella, diatoms, Scenedesmus, and Anabaena variabilis are relatively high.

[0066] In addition, the carbon dioxide absorption rate was measured. Carbon dioxide gas with the same concentration was introduced into the closed containers where each algal species was growing. After three days of aeration culture, the gas in each closed container was extracted, and the carbon dioxide content was measured by a gas chromatograph, so as to obtain the carbon dioxide absorption rates of different algal species at the same time as shown in Figure 2 It can be seen that from high to low are: Chlorella, Anabaena variabilis, Nostoc, Synechococcus, Scenedesmus.

[0067] Chlorella, Anabaena variabilis, and diatoms with better growth rates and carbon dioxide absorption rates were comprehensively selected for the adsorption experiment of modified nano - distillers' grains biochar, aiming to explore the growth situation and adsorption rate of microalgae on the modified nano - distillers' grains biochar, so as to evaluate the potential of the modified nano - distillers' grains biochar as a microalgae culture substrate. The specific test methods are as follows:

[0068] Take 10 g of the modified nano - distillers' grains biochar prepared in Example 1 and place it in an Erlenmeyer flask. Add the microalgae culture solution with a known concentration (2×10 7 -5×10 7 cells / mL), and record the initial microalgae concentration. Place the Erlenmeyer flask in a constant - temperature incubator, set the temperature at 25°C and the light intensity at 8000 lux, and conduct the culture. Measure the absorbance of the microalgae solution every two days to judge the growth situation of the microalgae. At the same time, measure the mass change of the modified nano - distillers' grains biochar from the beginning to the end to calculate the adsorption amount of the microalgae on the modified nano - distillers' grains biochar. The results are shown in Tables 6 - 8.

[0069] Adsorption effect of Chlorella on the modified nano - distillers' grains biochar in Table 6

[0070]

[0071] Table 7 Adsorption effect of diatoms on modified nano-distiller's grains biochar

[0072]

[0073] Table 8 Adsorption effect of Anabaena variabilis on modified nano-distiller's grains 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-distiller's grains biochar and exhibit a high adsorption rate. Further, it shows that the modified nano-distiller's grains biochar is a potential excellent microalgae culture substrate.

[0076] (2) Experiment on the selection of plant growth-promoting bacteria strains:

[0077] To prove that the plant growth-promoting bacteria strains selected in Examples 1-3 of the present invention have high biological characteristics and high carbon fixation as a comparative example, and to further evaluate the carbon fixation achieved through plant photosynthesis according to the promoting effect of the plant growth-promoting bacteria strains on plant growth, a variety of strains that may have the ability to promote plant growth are collected from natural environments such as soil, plant roots, and humus, and are preliminarily cultured. The collected strains are separated by the streak plate method and purified on LB medium to obtain single colonies. They are mixed and cultured with the screened microalgae strains, and the strains that can symbiose with microalgae are initially screened: numbered 1-4; and further physiological characteristics of the initially screened strains are determined, including the ability to produce indole-3-acetic acid (IAA), the ability to produce siderophores, the ability to dissolve phosphorus, etc., as shown in Table 9. These characteristics can reflect the promoting effect of the strains on plant growth. The screened strains are 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 optimal strain combination is determined.

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

[0079] Strain number IAA production (μg / mL) Siderophore production (μg / mL) Phosphate solubilizing ability (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 the physiological characteristics of strain 1 and strain 2 are relatively excellent, and they can be further subjected to a strain compounding experiment to explore their promotion rate on plant growth.

[0081] Table 10 Experimental comparison of the individual use and compound use of strain 1 and strain 2

[0082]

[0083] Among them: Growth promotion rate = (Growth amount of treatment group - Growth amount of control group) / Growth amount of control group × 100% (The average value of the growth promotion rates measured for root length, stem height, and dry weight respectively is taken).

[0084] The results show that the combination of strains 1 + 2 with the highest plant growth promotion rate can obtain the best plant growth promotion rate. Strains 1 and 2 were sequenced, and strain 1 was identified as Bacillus megaterium, and strain 2 was identified as Bacillus mucilaginosus. That is, the strain combination used in Examples 1 - 3 of the present invention is the best, and through subsequent experiments, the best effect can be obtained with a compounding ratio of 1:1 - 1.5:1.

[0085] (3) Experiment on selecting the mixing ratio of microalgae, plant growth - promoting bacteria, and modified nano - distiller's grains biochar:

[0086] Mixtures of microalgae suspension, plant growth - promoting bacteria suspension, and modified nano - distiller's grains biochar with different ratios were set. In each experimental group, the microalgae suspension with a cell count of 2×10 7 cells / mL and the plant growth - promoting bacteria suspension with a cell count of 1×10 6 cells / mL were mixed at a volume ratio of 2:1. The modified nano - distiller's grains biochar was added and mixed evenly according to the ratios set in Table 11, and then inoculated into the culture medium respectively. Each experimental group was cultured for one week under the conditions of 30°C and 8000 lux, and the growth conditions of microalgae and plant growth - promoting bacteria were recorded. At the same time, the carbon dioxide fixation efficiency of each experimental group was measured to evaluate the influence of different mixing ratios on the carbon reduction and carbon sequestration effects.

[0087] Experimental group 1: The ratio of modified nano - distiller's grains biochar added to the microalgae and plant growth - promoting bacteria suspension is 0.1 g / mL;

[0088] Experimental group 2: The ratio of modified nano - distiller's grains biochar added to the microalgae and plant growth - promoting bacteria suspension is 0.05 g / mL;

[0089] Experimental group 3: The ratio of modified nano - distiller's grains biochar added to the microalgae and plant growth - promoting bacteria suspension is 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 ratio of modified nano - distiller's grains biochar added to the microalgae and plant growth - promoting bacteria suspension is 0.1 - 0.05 g / mL, both the cell density of microalgae and the number of plant growth - promoting bacteria reach relatively high levels, and at the same time, the carbon dioxide capture efficiency is also the highest.

[0093] (4) Acidification modification comparison experiment

[0094] I. Material preparation: Take the modified distiller's grains biochar particles prepared in step (1) of Example 1. Using the same preparation method, the difference is that nitric acid is replaced with hydrochloric acid and sulfuric acid respectively to modify the properties of distiller's grains biochar, so as to verify and explore the influence mechanism of the unique chemical properties of nitric acid on the improvement of the properties of modified biochar. Set different acid concentration and treatment time gradients to obtain a series of modified biochar particle samples.

[0095] II. Performance characterization methods: Use specific surface area analysis (BET) to measure the specific surface area and pore structure of biochar; analyze the changes in surface functional groups of biochar by Fourier transform infrared spectroscopy (FTIR); observe the surface morphology of biochar using a scanning electron microscope (SEM); determine the contents of elements such as carbon, nitrogen, and oxygen in biochar with an elemental analyzer; use X-ray photoelectron spectroscopy (XPS) to analyze the chemical states of surface elements of biochar; and evaluate the adsorption capacity of biochar for specific pollutants (such as heavy metal ions or organic pollutants) through adsorption experiments.

[0096] III. Experimental results:

[0097] Table 12 Comparison of specific surface areas of modified biochar particles prepared by acid treatment at different concentrations

[0098]

[0099]

[0100] 1) Specific surface area and pore structure: Through the analysis of the test data results, the specific surface area of the distiller's grains biochar modified by nitric acid acidification increased significantly, the average pore diameter decreased, and the pore volume increased, and it was better than that of the biochar modified by hydrochloric acid and sulfuric acid. The specific surface area of the nitric acid-modified biochar could reach 30 m 2 / g, while those modified by hydrochloric acid and sulfuric acid were 23 m 2 / g and 28 m 2 / g respectively. 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 rearrangement of the carbon skeleton structure, promoting the further development and refinement of pores. While the oxidizing properties of hydrochloric acid and sulfuric acid are relatively weak, and the etching and structure rearrangement effects are not as obvious as those of nitric acid.

[0101] 2) Surface functional groups: FTIR spectra show that the content of oxygen-containing functional groups (such as carboxyl groups, hydroxyl groups, carbonyl groups, etc.) on the surface of nitric acid-modified biochar increases significantly, and the types of functional groups are more abundant. Compared with hydrochloric acid- and sulfuric acid-modified biochars, the absorption peak intensity of functional groups at specific wavenumbers of the nitric acid-modified sample is higher and the peak shape is sharper. The strong oxidizing property of nitric acid helps to introduce more reactive oxygen species on the surface of biochar, forming more types of oxygen-containing functional groups. The increase in these functional groups is beneficial 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 nitric acid-modified biochar increases, and nitrogen exists in multiple chemical states, including pyridine nitrogen, pyrrole nitrogen, and graphitic nitrogen, etc., among which the contents of pyridine nitrogen and pyrrole nitrogen with higher chemical activity are relatively high. In contrast, the effects of hydrochloric acid and sulfuric acid modification on the nitrogen content and nitrogen chemical state of biochar are relatively small. During the modification process, nitric acid can react with biochar through its nitrogen-containing groups, introducing nitrogen atoms into the biochar structure and changing the chemical environment of surface elements. This nitrogen doping effect enhances the surface activity and electron conduction ability of biochar, making it exhibit better performance in adsorption and catalysis, etc.

[0103] 4) Adsorption performance: The results of adsorption experiments show that the adsorption capacity of distiller's grains biochar modified by nitric acid acidification for target heavy metal ions (such as Pb 2 +, Cd 2 +, etc.) or organic pollutants (such as methylene blue, tetracycline, etc.) is significantly higher than that of hydrochloric acid- and sulfuric acid-modified biochars and raw biochar. For the adsorption of Pb 2 +, the removal rate of nitric acid-modified biochar can reach 93.82%, while those of hydrochloric acid- and sulfuric acid-modified biochars are 81.37% and 86.12% respectively, and that of raw biochar is only 48.33%.

[0104] Combined with the modification effects in terms of specific surface area, functional groups, and elemental composition mentioned above, the larger specific surface area of nitric acid-modified biochar provides more adsorption sites, and the rich oxygen-containing functional groups and nitrogen doping effect enhance its affinity and chemical adsorption for pollutants, thus significantly improving its adsorption performance. Due to the relatively weak modification effects in these aspects, the adsorption performance improvement of hydrochloric acid- and sulfuric acid-modified biochars is not as significant as that of nitric acid-modified biochar. The distiller's grains biochar modified by nitric acid acidification has been significantly improved in terms of specific surface area, surface functional groups, elemental composition, and adsorption performance compared with hydrochloric acid and other conventionally acidified biochars. This improvement is mainly attributed to the unique strong oxidizing property, nitrogen doping ability of nitric acid, and its deep modification effect on the surface and structure of biochar. Therefore, using nitric acid to modify distiller's grains biochar enables the modified distiller's grains biochar to have a broader application prospect in the fields of agriculture, environmental remediation, etc.

[0105] Application Example 5: Carbon Sequestration Efficiency

[0106] Place the nano-porous distiller's grains biochar loaded with bioactive materials (1 kg) prepared in Example 1 in a closed reactor, and place the reactor under the conditions of 28 °C and 8000 lux for cultivation. Monitor the change in carbon dioxide concentration in the reactor every two days to judge the carbon dioxide absorption rate of the nano-porous distiller's grains biochar loaded with bioactive materials, as shown in Table 13.

[0107] Table 13 Carbon Dioxide Absorption Rate of Nano-porous Distiller's Grains Biochar Loaded with Bioactive Materials

[0108] Time (days) 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] As can be seen from Table 13, the nano-porous distiller's grains biochar loaded with bioactive materials prepared by the present invention has good carbon sequestration ability.

[0110] Application Example 6: Promote Plant Growth

[0111] Mix the nano-porous distiller's grains biochar loaded with bioactive materials prepared in Example 1 with soil, and the mixing weight ratio is 1:3. Plant tomatoes in it, select tomato seeds of the same excellent quality, and set up an experimental group and a control group (without adding nano-porous distiller's grains biochar loaded with bioactive materials) at the same time. Regularly observe and record the growth of tomatoes, including plant height and leaf area, and the records are shown in Table 14.

[0112] Table 14 Growth of Tomatoes Planted with Nano-porous Distiller's Grains Biochar Loaded with Bioactive Materials

[0113]

[0114] From the comparison data in Table 14, it can be seen that the experimental group adding nano-porous distiller's grains biochar loaded with bioactive materials has obvious advantages in both plant height and leaf area. Please refer to Figure 4 (experimental group) and Figure 5 (control group) shown in the pictures of the growth of tomatoes on the 20th day. It can also be seen that the soil adding the nano-porous distiller's grains biochar loaded with bioactive materials prepared in Example 1 of the present invention has an obvious promoting effect on the growth of tomatoes.

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

[0116] Select cherry tomatoes and cucumbers, take cherry tomatoes and cucumbers with similar growth conditions as a group, divide them into three groups in total, and set two replicate samples for each group. Uniformly spray the same concentration of pesticides on the three groups of fruits and vegetables to simulate the pesticide use in actual production. Place the fruits and vegetables outdoors under natural conditions for 1 month.

[0117] 1) Irrigation treatment

[0118] A) Blank control group: Irrigate with water according to the conventional method, 500 mL each time, and irrigate at regular intervals according to the weather conditions to keep the soil humidity appropriate.

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

[0120] C) Experimental group 2: Mix and irrigate with the nano-porous distillers grains biochar loaded with bioactive materials prepared in Example 1 of the present invention and water-soluble fertilizer containing trace elements. Add 10 g of nano-porous distillers grains biochar loaded with bioactive materials to 100 mL of water-soluble fertilizer containing trace elements and 500 mL of water, and the irrigation frequency is the same as that of the control group and control group 1.

[0121] D) Detection of agricultural residues

[0122] After 25 days, randomly select multiple fruit and vegetable samples from the three groups respectively, and detect the residual amounts of organochlorine and organophosphorus pesticides in the fruits and vegetables. Record the detection results of each sample and calculate the average agricultural residue content of each group.

[0123] Table 15 Influence of different treatments on agricultural residues in fruits and vegetables

[0124]

[0125] It can be seen from the experimental results in Table 15 that the mixture of the nano-porous distillers grains biochar loaded with bioactive materials prepared in the present invention and water-soluble fertilizer containing trace elements can effectively reduce the agricultural residues in fruits and vegetables.

Claims

1. A preparation method of a nano-porous distiller's grains carbon bioactive material, comprising the following steps: (1) Mix the microalgae suspension and the plant growth-promoting bacteria suspension at a volume ratio of 1.5:1 - 3:1, and then add them to the modified distiller's grains biochar particles with a size of 0.5 - 2 mm. Gently stir to make the microorganisms evenly adsorbed on the surface and pores of the modified distiller's grains biochar; the addition ratio of the modified distiller's grains biochar particles in the mixed suspension is 0.05 - 0.1 g / mL; the surface area of the modified distiller's grains biochar particles is 25 - 35 m 2 / g, and the average adsorption pore diameter is 2.5 - 3.5 nm; the microalgae selected for the microalgae suspension are one of Chlorella vulgaris, Anabaena variabilis, and Nostoc sp.; the plant growth-promoting bacteria selected for the plant growth-promoting bacteria suspension are a mixture of Bacillus megaterium and Bacillus mucilaginosus with a mass ratio of 1:1 - 1.5:1; (2) Under static conditions, set the temperature to 20 - 30 °C, the light intensity to 5000 - 10000 Lux, and maintain the pH value at 7.0 - 8.0, allowing microorganisms to naturally penetrate and adhere to the inside of the modified distiller's grains biochar for 24 - 48 h, and adjust the moisture content of the final product to be between 30 - 40% to obtain a bioactive material of a nano-porous distiller's grains carbon bioactive material loaded with microalgae and growth-promoting bacteria; The preparation method of the modified distiller's grains biochar particles uses distiller's grains as raw materials, conducts high-temperature pyrolysis under anaerobic conditions, and then conducts nitric acid modification to obtain modified distiller's grains biochar particles with a porous structure and good adsorption performance, specifically including the following steps: 1) Preliminarily screen the distiller's grains to remove impurities and large pieces of matter therein; 2) Dry the screened distiller's grains at 103 - 107 °C until constant weight; 3) Conduct high-temperature pyrolysis on the dried distiller's grains in an anaerobic environment at a pyrolysis temperature of 800 °C; after pyrolysis is completed, naturally cool to room temperature to obtain carbonized distiller's grains biochar; 4) Mix the obtained distiller's grains biochar with a nitric acid solution to conduct acid modification on the distiller's grains biochar; 5) Grind the acid-modified distiller's grains biochar into powder and sieve it to obtain particles with a particle size of 0.5 - 2 mm as a carrier for loading microalgae and growth-promoting bacteria.

2. The preparation method of the nano-porous distiller's grains bioactive carbon material according to claim 1, wherein In step (2), the control of the water content is achieved by natural air drying or slight heating.

3. The preparation method of the nano-porous distiller's grains bioactive carbon material according to claim 1, 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.

4. The preparation method of the nano-porous distiller's grains bioactive carbon material according to claim 1, characterized in that: The specific preparation method of the plant growth-promoting bacteria suspension described in step (1) is as follows: Select the sources of pure and pollution-free Bacillus megaterium and Bacillus mucilaginosus, use LB medium, and culture at a pH of 7.0, a temperature of 30 - 37 °C, and a shaker speed of 200 r / min until the number of bacterial cells grows to 1×10 6 - 5×10 6 cells / mL; Centrifuge or filter to collect the bacterial cells, remove the supernatant, and then resuspend the bacterial cells with a buffer solution, and adjust the suspension concentration to 1×10 6 cells / mL for use. After sufficient stirring or shaking to make it uniform, the Bacillus suspension is obtained; The whole preparation process is carried out under sterile conditions.

5. The preparation method of the nano-porous distiller's grains bioactive carbon material according to claim 1, wherein: The inoculation amount of microalgae in the microalgae suspension described in step (1) is 2×10 7 -5×10 7 cells per mL, and the inoculation amount of plant growth-promoting bacteria in the plant growth-promoting bacteria suspension is 1×10 6 -5×10 6 cells per mL.

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