Chitosan-loaded biochar and applications thereof

Abstract

This invention provides a chitosan-loaded biochar and its applications. The provided chitosan-loaded biochar is prepared by treating biochar with an alkaline solution, followed by mixing the modified alkaline-treated biochar with a solution containing dissolved chitosan. The chitosan-loaded biochar prepared by this invention can be used to adsorb chemical pollutants in aquatic environments and can also be used as a carrier for microorganisms. By modifying the biochar with potassium hydroxide, this invention allows for the loading of more chitosan, significantly improving the utilization rate of chitosan. This invention increases the specific surface area of ​​the biochar, thereby increasing the attachment sites for chitosan. Compared to other methods that increase chitosan loading by increasing the thickness of the chitosan, this invention not only allows for a greater chitosan loading on the biochar but also ensures a more uniform distribution of chitosan on the biochar, fully leveraging the advantages of chitosan-loaded biochar.

Description

Technical Field

[0001] This invention belongs to the field of biochar technology, specifically relating to a chitosan-supported biochar and its applications. Background Technology

[0002] Biochar is a carbon-rich substance mainly produced by high-temperature pyrolysis of agricultural biomass waste under low-oxygen or anaerobic conditions. Its preparation requires low energy consumption, has a wide range of raw material sources, and low production costs. The application of biochar as an adsorbent in pollution control has received significant attention from governments and relevant scholars. After specific physical or chemical modifications, raw biochar can achieve a substantial increase in its adsorption capacity for specific pollutants, making it an effective means to promote the application of biochar in environmental pollution control.

[0003] Chitosan is a non-toxic and pollution-free polymer containing numerous functional groups (such as amino and carboxyl groups) closely related to its adsorption properties. It can bind to target molecules and adsorb pollutants through hydrogen bonding and complexation. Powdered chitosan dissolves into a gel state, which enhances its adsorption capacity, but it is difficult to recycle and tends to clump together when used alone, leading to reduced adsorption efficiency. Therefore, loading chitosan onto the surface of biochar to create a chitosan-biochar composite material can enhance the adsorption capacity of biochar through chitosan modification while overcoming the limitations of using chitosan alone, thus leveraging the synergistic advantages of both.

[0004] However, in current production practices, chitosan biochar generally suffers from low chitosan loading efficiency on the biochar surface, resulting in insufficient pollutant removal and a significant waste of chitosan resources, greatly limiting its application. Current research attempts to increase chitosan loading by enhancing the degree of carboxylation on the biochar surface and increasing the chitosan loading thickness; however, this method has proven ineffective in pollutant removal. This is because it fails to fundamentally address the problem of uneven chitosan loading on the biochar surface and the insufficient effective binding sites between the complex and pollutants. Summary of the Invention

[0005] This invention addresses the problems of low chitosan loading efficiency and uneven loading distribution in existing biochar, and provides a chitosan-loaded biochar and its applications.

[0006] The chitosan-loaded biochar provided by the present invention is prepared by treating biochar with an alkaline solution, and then mixing the alkaline-treated modified biochar with a solution containing chitosan.

[0007] The alkaline solution mentioned is an alkaline solution with a pH of 11 to 14;

[0008] As a specific example, the alkaline solution is a potassium hydroxide solution with a concentration of 0.5 to 1.5 mol / L;

[0009] The chitosan-loaded biochar prepared by this invention can be used to adsorb chemical pollutants in aquatic environments;

[0010] The pollutant mentioned, as a specific example, is methylene blue;

[0011] The chitosan-loaded biochar provided by this invention can also be used as a carrier for microorganisms to promote their function.

[0012] The microorganism described, as a specific example, is Acinetobacter pittii MAP2303, which has a strong phosphate-solubilizing ability. This strain was deposited at the China Center for Type Culture Collection of Wuhan University on May 17, 2023, with the biological accession number CCTCC NO:M 2023778.

[0013] In another aspect, the present invention provides a microbial solid inoculant, which is biochar loaded with chitosan carrying the aforementioned Acinetobacter pituitaria MAP2303.

[0014] This invention, by modifying biochar with potassium hydroxide, enables it to support a greater amount of chitosan, significantly improving chitosan utilization. By increasing the specific surface area of ​​the biochar, this invention increases the attachment sites for chitosan. Compared to other methods that increase chitosan loading by increasing chitosan thickness, this invention not only allows for greater chitosan loading on the biochar but also ensures a more uniform distribution of chitosan on the biochar, thus fully leveraging the advantages of chitosan biochar. Attached Figure Description

[0015] Figure 1 This invention provides a method for increasing the chitosan loading on the surface of biochar and a flowchart of the adsorption of methylene blue.

[0016] Figure 2 This is a scanning electron microscope image of the chitosan-supported biochar product prepared in Example 1 of this invention;

[0017] Figure 3 This is a schematic diagram (X-ray diffraction diagram) of the chitosan biochar (CHKBC) prepared in Example 1 of the present invention and the chitosan biochar (CHBC) of the comparative example, showing the chitosan loading efficiency. CHKBC is the chitosan biochar prepared in Example 1; CHBC is the chitosan biochar prepared in Comparative Example 1; BC is the unmodified biochar prepared in Comparative Example 2; and Chitosan is chitosan.

[0018] Figure 4 This is a schematic diagram (thermogravimetric analysis diagram) of the chitosan biochar (CHKBC) prepared in Example 1 of the present invention and the chitosan biochar (CHBC) of Comparative Example 1 loaded with chitosan.

[0019] Figure 5 This is a schematic diagram showing the methylene blue removal efficiency results of chitosan biochar (CHKBC) prepared in Example 1 and chitosan biochar (CHBC) in Comparative Example 1.

[0020] Figure 6 These are the fitting curves of the adsorption kinetics of methylene blue on the chitosan biochar (CHKBC) prepared in Example 1 and the chitosan biochar (CHBC) in Comparative Example 1.

[0021] Figure 7 These are adsorption isotherms of methylene blue on chitosan biochar (CHKBC) prepared in Example 1 and chitosan biochar (CHBC) in Comparative Example 1.

[0022] Figure 8 These are microscopic images of Acinetobacter pitera screened in Example 5 of this invention;

[0023] Figure 9 This is a photograph of the colonies of Acinetobacter piezoides screened in Example 5 of the present invention on beef extract peptone medium;

[0024] Figure 10 This is a graph showing the effect of chitosan biochar on the viable count of Acinetobacter piezoides;

[0025] Figure 11 This is a graph showing the effect of chitosan biochar on the phosphorus-solubilizing ability of Acinetobacter piezoides. Detailed Implementation

[0026] The biochar provided by this invention increases the chitosan attachment sites on the surface of the biochar, thereby increasing the chitosan loading on the biochar surface. It also effectively promotes the probability of chitosan contacting pollutants, thereby improving its adsorption efficiency for water pollutants and the loading of beneficial bacteria.

[0027] The preparation method of the chitosan-supported biochar provided by this invention is as follows:

[0028] Step 1: After drying the rice straw, crush and sieve it to obtain the prepared biomass, which is then pyrolyzed to obtain biochar. Alternatively, after drying the rice straw, pass it through an 80-mesh sieve to obtain the prepared biomass. Place the biomass in a muffle furnace for pyrolysis, raising the temperature to 500℃ at a rate of 10℃ / min, maintaining it for 1 hour, and then cooling it to room temperature to obtain biochar.

[0029] Rice straw can be replaced with agricultural waste such as rice husks, corn stalks, and peanut shells, or other raw materials used to prepare biochar.

[0030] Correspondingly, existing methods can also be used to prepare biochar. For example, crushed rice straw can be passed through an 80-mesh sieve and heated to 500°C in a muffle furnace at a heating rate of 10°C / min, held for 1 hour, and then cooled to room temperature to obtain biochar. Another example is to pass crushed rice straw through an 80-mesh sieve and place it in a nitrogen-filled tube furnace, heat it to 500°C at a heating rate of 15°C / min, hold for 1 hour, and then cool to room temperature to obtain biochar.

[0031] Step 2: Prepare a potassium hydroxide solution, add biochar and stir with a magnetic stirrer, let stand, then wash repeatedly with distilled water until the pH no longer changes to ensure that all residual KOH is removed. Finally, dry at 60°C, pulverize and pass through an 80-mesh sieve to obtain potassium hydroxide modified biochar.

[0032] The concentration of the potassium hydroxide solution is 0.5–1.5 mol / L;

[0033] Other alkaline solutions, such as sodium hydroxide or calcium hydroxide, can also be used, but the final product prepared is not as effective as that prepared with potassium hydroxide.

[0034] Step 3: Dissolve chitosan in an acidic solution with a degree of deacetylation of over 75%, and stir magnetically until completely dissolved;

[0035] Step 4: Mix the potassium hydroxide-modified biochar with the above chitosan solution and stir, then ultrasonically mix until homogeneous; add sodium hydroxide solution to adjust the pH to 12, continue ultrasonic mixing until homogeneous, and let stand; after standing, filter, wash, dry, grind, and sieve the obtained product to obtain chitosan-loaded biochar.

[0036] The chitosan-loaded biochar prepared in this invention can be used as an adsorbent to adsorb pollutants in water, or as a carrier for bacterial strain attachment to prepare immobilized bacterial agents.

[0037] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0038] Example 1: Preparation of chitosan-loaded biochar

[0039] The method for preparing chitosan-loaded biochar in this embodiment is as follows: Figure 1 As shown, the specific steps include:

[0040] After drying rice straw in an oven at 60℃ for 24 hours, it was crushed using a pulverizer and passed through an 80-mesh sieve to obtain biomass for later use. The biomass was then placed in a muffle furnace for pyrolysis, heated to 500℃ at a rate of 10℃ / min, held for 1 hour, and then cooled to room temperature in the furnace to obtain biochar.

[0041] Weigh 50g of the above biochar, add 500mL of KOH (1mol / L) solution, stir in a magnetic stirrer for 60min, let stand for 3h to ensure sufficient reaction, then wash repeatedly with distilled water until the pH no longer changes to ensure that all residual KOH is removed, and finally dry at 60℃, pulverize and pass through an 80-mesh sieve to obtain potassium hydroxide modified biochar.

[0042] 1 g of chitosan was dissolved in 200 mL of CH3COOH (2% v / v) and stirred with a magnetic stirrer until completely dissolved. Then, 2 g of potassium hydroxide-modified biochar was added, and stirring was continued for 60 min. Next, NaOH (1 mol / L) solution was added to adjust the pH to 12. The mixture was then placed in an ultrasonic oscillator for 60 min to ensure homogeneity. The mixture was then sealed and allowed to stand for 24 h. The precipitate was separated and washed with distilled water until the pH no longer changed. Finally, it was dried at 60 °C. Rice husks were dried in an oven at 60 °C for 24 h, then pulverized and passed through an 80-mesh sieve to obtain biomass. The biomass was then pyrolyzed in a muffle furnace at a heating rate of 10 °C / min to 500 °C and held for 1 h. After cooling to room temperature in the furnace, biochar was obtained. This biochar was then pulverized and passed through an 80-mesh sieve to obtain the chitosan biochar prepared by the method of this invention.

[0043] Example 2: Preparation of chitosan-loaded biochar

[0044] After drying rice husks in an oven at 60℃ for 24 hours, they are crushed using a pulverizer and passed through an 80-mesh sieve to obtain biomass for later use. The biomass is then placed in a muffle furnace for pyrolysis, heated to 500℃ at a rate of 10℃ / min, held for 1 hour, and then cooled to room temperature in the furnace to obtain biochar.

[0045] Weigh 50g of the above biochar, add 500mL of KOH (0.5mol / L) solution, stir in a magnetic stirrer for 90min, let stand for 3h to ensure sufficient reaction, then wash repeatedly with distilled water until the pH no longer changes to ensure that all residual KOH is removed, and finally dry at 60℃, pulverize and pass through an 80-mesh sieve to obtain potassium hydroxide modified biochar.

[0046] Dissolve 1–3 g of chitosan in 200 mL of CH3COOH (2%, v / v) and stir with a magnetic stirrer until completely dissolved. Then add 2 g of potassium hydroxide-modified biochar and continue stirring for 60 min. Next, add NaOH (1 mol / L) solution to adjust the pH to 12. Place the mixture in an ultrasonic oscillator for 60 min to ensure homogeneity. Then, seal the mixture and let it stand for 24 h. Separate the precipitate and wash it with distilled water until the pH no longer changes. Finally, dry it at 60 °C and pulverize it through an 80-mesh sieve to obtain the chitosan biochar prepared by the method of this invention.

[0047] Comparative Example 1: Existing ordinary chitosan biochar

[0048] The preparation method includes the following steps:

[0049] Rice straw was dried in an oven at 60℃ for 24 hours, then pulverized and passed through an 80-mesh sieve to obtain biomass. The biomass was then pyrolyzed in a muffle furnace at a heating rate of 10℃ / min to 500℃ and held for 1 hour. After cooling to room temperature in the furnace, biochar was obtained. 1 g of chitosan was dissolved in 200 mL of CH3COOH (2%, v / v) and stirred with a magnetic stirrer until completely dissolved. Then, 2 g of biochar was added and stirring was continued for 1 hour. After that, NaOH (1 mol / L) solution was added to adjust the pH to 12. The mixture was placed in an ultrasonic oscillator for 1 hour to ensure homogeneity. Subsequently, the mixture was sealed and allowed to stand for 24 hours. The precipitate was separated and washed with distilled water until the pH no longer changed. Finally, it was dried at 60℃, pulverized, and passed through an 80-mesh sieve to obtain ordinary chitosan biochar.

[0050] Comparative Example 2: Unmodified biochar

[0051] The method for preparing raw, unmodified biochar includes the following steps:

[0052] After drying rice straw in an oven at 60℃ for 24 hours, it was crushed using a pulverizer and passed through an 80-mesh sieve to obtain biomass for later use. The biomass was then placed in a muffle furnace for pyrolysis, heated to 500℃ at a rate of 10℃ / min, held for 1 hour, and then cooled to room temperature in the furnace to obtain biochar.

[0053] Example 3: Physicochemical property analysis of chitosan-supported biochar

[0054] Taking Example 1 as an example, the image and microstructure diagram of the prepared chitosan-supported biochar product are as follows: Figure 2 As shown, although the surface of the biochar is coated with chitosan, it still exhibits a large number of pore structures. This indicates that the chitosan biochar product provided by this invention can increase its contact surface with pollutants and improve adsorption efficiency when used as an adsorbent.

[0055] Taking the chitosan-loaded biochar prepared in Example 1 as an example, we studied its chitosan loading capacity.

[0056] 1. Example 1 and Comparative Biochar X-ray Diffraction (XRD) Analysis Experiment

[0057] Take 15 mg each of the chitosan biochar (CHKBC) prepared in Example 1, the ordinary chitosan biochar (CHBC) prepared in Comparative Example 1, the original biochar (BC) prepared in Comparative Example 2, and chitosan, and place them in a glass sample holder. Press them flat with frosted glass, place them on the sample stage, set the angle range to 10–80°, and the testing rate to 2° / min. The test results are as follows: Figure 3 As shown, Chitosan exhibits a special peak at 20°, which is the peak of chitosan crystals. CHKBC and CHBC also have obvious stretching peaks at this point, proving that the chitosan loading was successful. Moreover, the peak at this point is stronger for CHKBC, the chitosan biochar prepared in Example 1, than for CHBC, indicating that the chitosan diffraction intensity on CHKBC is higher and more chitosan is exposed.

[0058] 2. Thermogravimetric analysis of biochar in Example 1 and the comparative example

[0059] Ten mg each of the chitosan biochar (CHKBC) prepared in Example 1, the ordinary chitosan biochar (CHBC) prepared in Comparative Example 1, and the original biochar (BC) prepared in Comparative Example 2 were added to a thermogravimetric analysis balance. The thermogravimetric analyzer was set to a heating range of 30–800 °C and a heating rate of 10 °C / min. The experimental results are as follows: Figure 4 As shown, the weight of the chitosan biochar CHKBC prepared by the method of the present invention decreased by 40.49%, while the weight of the comparative chitosan biochar CHBC decreased by 27.41%. The weight loss of the chitosan biochar prepared by the method of the present invention is more significant, which is due to the large-scale decomposition of chitosan in the range of 240-330℃. This proves that the CHKBC prepared by the method of the present invention has a higher chitosan loading than CHBC.

[0060] Example 4: Performance in removing methylene blue

[0061] Taking the chitosan biochar prepared in Example 1 as an example, this invention illustrates the performance of the chitosan biochar CHKBC prepared in Example 1 in removing methylene blue.

[0062] 1. Test on the efficiency of chitosan biochar (CHKBC) in removing methylene blue

[0063] Weigh 100 mg of methylene blue powder, dissolve it, and dilute to 1000 mL to prepare a 100 mg / L methylene blue solution. Add 0.5–3 g / L of each of Example 1 (CHKBC), Comparative Example 1 (CHBC), and Comparative Example 2 (BC) to the methylene blue solution. Shake the mixture at 180 rpm for 8 hours. Filter the sample using a 0.45 μm syringe filter. Measure the residual methylene blue in the supernatant using a UV spectrophotometer. The adsorption amount of methylene blue, Q, is determined. e The formula for calculating the removal rate η is as follows:

[0064]

[0065]

[0066] Q e The equilibrium adsorption capacity of biochar for methylene blue; CO and C e The values ​​represent the methylene blue concentrations before and after adsorption equilibrium, respectively; V is the solution volume; m is the amount of biochar added; and η is the removal rate of methylene blue by biochar.

[0067] The test results are as follows Figure 5 As shown, the chitosan biochar (CHKBC) prepared in Example 1 exhibited greater adsorption capacity than the ordinary chitosan biochar prepared in Comparative Example 1 and the original biochar prepared in Comparative Example 2 at different addition amounts. When the addition amount was 3 g / L, CHKBC achieved a removal rate of 99.90% for methylene blue at a concentration of 100 mg / L, while CHBC only achieved 55.79%. Therefore, it can be concluded that the chitosan biochar prepared by the method of the present invention has a high removal efficiency for methylene blue.

[0068] 2. Adsorption kinetics of methylene blue removal by chitosan biochar (CHKBC)

[0069] Take 20 mL of a 100 mg / L methylene blue solution and add 0.04 g of BC, CHBC, and CHKBC (addition standard 2 g / L), respectively. Place in a constant temperature (25℃) shaker and take samples at 5 min, 10 min, 20 min, 30 min, 40 min, 60 min, 120 min, 180 min, 300 min, 600 min, 960 min, and 1440 min. Filter the solution using a 0.45 μm syringe filter (organic system) and determine the solution concentration; calculate the remaining content. Use pseudo-first-order and pseudo-second-order kinetic models to describe the adsorption kinetic data and explore the adsorption capacity of each adsorbent surface for methylene blue. The kinetic model equations are expressed as follows:

[0070] Pseudo-first-order dynamic equation:

[0071] ln(Qe -Q t )=lnQ e –K1t

[0072] Pseudo-second-order dynamic equations:

[0073]

[0074] Q e and Q t K1 (g / mg / min) and K2 (g / mg / min) are the adsorption amounts of MB on the adsorbent at equilibrium and at different times, respectively; K1 (g / min) and K2 (g / mg / min) are the rate constants of the pseudo-first-order and pseudo-second-order models.

[0075] Figure 6 Table 1 shows the adsorption kinetic curves and fitting parameters of methylene blue for Example 1, Comparative Example 1, and Comparative Example 2. The results show that the chitosan biochar prepared in Example 1 completed 96.96% adsorption within 60 min, while the ordinary chitosan biochar in Comparative Example 1 completed 95.00%. The original biochar in Comparative Example 2 completed 91.25%. Both the chitosan biochar of Example 1 and the ordinary chitosan biochar of Comparative Example 1 conform to the pseudo-second-order kinetic model, indicating that chitosan mainly acts as a physical adsorption site for adsorbing methylene blue.

[0076] Table 1: Adsorption Kinetics Model Parameters

[0077]

[0078] 3. Isothermal adsorption test of chitosan biochar (CHKBC) for removing methylene blue

[0079] 20 mL of methylene blue solutions with initial concentrations of 10 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, and 500 mg / L were taken, and 0.04 g of BC, CHBC, and CHKBC were added respectively. The solutions were then shaken in a constant temperature (25℃) shaker for 24 h. After filtration through a 0.45 μm syringe filter (organic system), the solution concentrations were measured, and the remaining methylene blue content was calculated. The Langmuir and Freundlich isotherm models were used to simulate the adsorption of methylene blue by biochar, and their expressions are as follows:

[0080] Langmuir model:

[0081]

[0082] Freundlich model:

[0083]

[0084] Where C e Q represents the concentration of MB at adsorption equilibrium (mg / L); e The equilibrium amount of MB adsorbed on the surface of biochar (mg·g) -1 ), Q m Maximum adsorption capacity of biochar for methylene blue (mg / g); K L (L / mg) and K F (mg 1-N / L N ·g) are Langmuir constant and Freundlich constant, respectively; N is the adsorption strength constant.

[0085] Figure 7 Table 2 shows the isothermal adsorption model fitting curves and fitting parameters for methylene blue in Example 1, Comparative Example 1, and Comparative Example 2. According to the fitting results, the maximum adsorption capacity of the chitosan biochar prepared in Example 1 for methylene blue is 51.07 mg / g, while the maximum adsorption capacity of the ordinary chitosan biochar prepared in Comparative Example 1 is 25.13 mg / g. This indicates that the ability of the chitosan biochar prepared in this invention to adsorb methylene blue is greatly improved compared with that of the ordinary chitosan biochar in Comparative Example 1.

[0086] Table 2: Parameters of the Isothermal Adsorption Model

[0087]

[0088] Example 5: Application of chitosan biochar as a carrier in the preparation of solid bacterial preparations

[0089] 1. Screening and determination of taxonomic position of strains

[0090] 1) Screening methods for phosphate-solubilizing strains

[0091] Add 1g of rice rhizosphere soil to 9mL of sterile water, shake in a constant temperature shaker at 30℃ and 180r / min for 30min, and let stand for 10min to obtain a soil suspension. Dilute 10... -3 -10 -7 After dilution, 100 μL was evenly spread on beef extract peptone solid medium and cultured for 1-3 days. Bacterial colony growth was observed, and colonies were repeatedly streaked for purification to obtain a single strain. The strain was then spot-inoculated on Ca3(PO4)2 solid medium and cultured at 30℃ for 3-7 days. The presence of a phosphate-solubilizing zone around the colony was observed, and the colony diameter (d) and the clear zone diameter (D) were measured. The phosphate solubilization coefficient (D / d) was calculated, and bacteria with highly efficient phosphate solubilization effects were screened.

[0092] 2) Determination of the taxonomic status of phosphate-solubilizing strains

[0093] Gram staining and microscopic observation were performed by spreading the culture medium onto a smear, and then adding crystal violet staining solution, Gram's iodine solution, decolorizing solution, and safranin counterstaining solution in sequence, followed by microscopic observation.

[0094] DNA was extracted from phosphate-solubilizing bacteria using the Ezup column-based bacterial genomic DNA extraction kit. PCR amplification, reaction system settings, cycling conditions, gel electrophoresis, and final purification and sequencing were performed sequentially, yielding a 1446 bp PCR product. The obtained 16S rDNA sequence (SEQ ID NO: 1) was compared with the ribosome database http: / / rdp.cme.msu.edu / index.jsp. Identification revealed that this strain shared over 99% similarity with several different species of the Acinetobacter genus, and the strain was identified as Acinetobacter pittii MAP2303.

[0095] Microscopic observation and Gram staining revealed that *Acinetobacter piezophyllum* was present in coccobacillus-like, double, and singly forms, and was Gram-negative. Figure 8 The colonies are round, grayish-white, moist, smooth, and have neat edges. Figure 9 ).

[0096] The 16S rDNA sequence is as follows:

[0097] TTTGATCCAGGTCAGATTGAACGCTGGCGGCAGGCTTAACA

[0098] CATGCAAGTCGAGCGGAGAGAGGTAGCTTGCTACTGATCTTAGCG

[0099] GCGGACGGGTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGA

[0100] CAACATTTCGAAAGGAATGCTAATACCGCATACGTCCTACGGGAG

[0101] AAAGCAGGGGATCTTCGGACCTTGCGCTAATAGATGAGCCTAAGT

[0102] CGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATC

[0103] TGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACA

[0104] CGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAA

[0105] TGGGCGCAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGC

[0106] CTTATGGTTGTAAAGCACTTTAAGCGAGGAGGAGGCTACTTTAGAT

[0107] AATACCTAGAGATAGTGGACGTTACTCGCAGAATAAGCACCGGCT

[0108] AACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTA

[0109] ATCGGATTTACTGGGCGTAAAGCGCGCGTAGGCGGCTAATTAAGT

[0110] CAAATGTGAAATCCCCGAGCTTAACTTGGGAATTGCATTCGATACT

[0111] GGTTAGCTAGAGTGTGGGAGAGGATGGTAGAATTCCAGGTGTAGC

[0112] GGTGAAATGCGTAGAGATCTGGAGGAATACCGATGGCGAAGGCA

[0113] GCCATCTGGCCTAACACTGACGCTGAGGTGCGAAAGCATGGGGA

[0114] GCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGTC

[0115] TACTAGCCGTTGGGGCCTTTGAGGCTTTAGTGGCGCAGCTAACGC

[0116] GATAAGTAGACCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCA

[0117] AATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTA

[0118] ATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATAGTAAG

[0119] AACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTTACATACAGG

[0120] TGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAA

[0121] GTCCCGCAACGAGCGCAACCCTTTTCCTTATTTGCCAGCGAGTAAT

[0122] GTCGGGAACTTTAAGGATACTGCCAGTGACAAACTGGAGGAAGG

[0123] CGGGGACGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACA

[0124] CACGTGCTACAATGGTCGGTACAAAGGGTTGCTACCTAGCGATAG

[0125] GATGCTAATCTCAAAAAGCCGATCGTAGTCCGGATTGGAGTCTGC

[0126] AACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGA

[0127] ATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTC

[0128] ACACCATGGGAGTTTGTTGCACCAGAAGTAGCTAGCCTAACTGCA

[0129] AAGAGGGCG。

[0130] 2. Phosphate-solubilizing effect of Acinetobacter pittii MAP2303

[0131] Dissolve 3g beef extract, 10g peptone, and 5g sodium chloride in 1000mL of deionized water, adjust the pH to 7.0–7.2, and autoclave at 121℃ for 20min to prepare beef extract peptone medium. Add 1mL of Acinetobacter piezocarp cryopreservation solution to 99mL of beef extract peptone liquid medium and incubate at 30℃ with shaking at 180r / min for 20h until the logarithmic growth phase, to prepare Acinetobacter piezocarp suspension. Add 1mL of the logarithmic growth phase Acinetobacter piezocarp suspension to 99mL of liquid medium containing 5g / L Ca3(PO4)2 and incubate for 1–7 days, then determine the soluble phosphorus content.

[0132] The cultured carbon-bacterial composite solution was centrifuged at 4℃ and 6000 r / min for 15 min. 0.2 mL of the supernatant was taken and added dropwise to a 50 mL volumetric flask. 30 mL of H2O, dinitrophenol indicator, 2 mol / L NaOH solution, 1 mol / L H2SO4 solution, and 5 mL of molybdenum antimony anti-colorimetric reagent were added sequentially. Finally, water was added to bring the volume to 50 mL. After shaking, the solution was allowed to stand for 30 min. The blank control solution was used as a reference for zeroing. The soluble phosphorus content in the culture medium was determined by spectrophotometry at a wavelength of 880 nm. Each treatment was repeated three times.

[0133] The results showed that the soluble phosphorus content of Acinetobacter pituitariae MAP2303 screened in this invention reached 213.21 mg / L on the 3rd day of culture, which was significantly higher than that of other phosphate-solubilizing bacteria.

[0134] 3. Preparation of solid inoculum of Acinetobacter piezocarpa supported on chitosan biochar

[0135] A single colony of *Acinetobacter pitride* was picked and added to 100 mL of beef extract peptone broth, and cultured on a shaker at 180 rpm at 30°C for 20 h until the logarithmic growth phase. Biochar was added to the beef extract peptone broth at a ratio of 3% (g:v) and autoclaved. Then, *Acinetobacter pitride* culture was added to the beef extract peptone broth at a ratio of 1% (v:v). The mixture was incubated at 30°C and 180 rpm for 3 days. After incubation, the culture was centrifuged to obtain a solid bacterial preparation.

[0136] 4. Application and effects of the prepared chitosan biochar-supported Acinetobacter piezoides solid inoculum

[0137] Figure 11As shown, the number of viable Acinetobacter pituitaria in the solid bacterial agent prepared by loading Acinetobacter pituitaria with chitosan biochar CHKBC of the present invention was significantly increased by 102.84% compared with the number of viable free bacteria. In contrast, the number of viable Acinetobacter pituitaria in the solid bacterial agent prepared by Comparative Example 2BC was significantly increased by 159.84%. This indicates that the chitosan biochar prepared by the present invention can serve as an excellent carrier for Acinetobacter pituitaria MAP2303, with significantly better effects than immobilized bacterial agents prepared by ordinary biochar. It can provide a safe habitat for MAP2303 and promote the growth and reproduction of MAP2303.

[0138] The results showed that the solid bacterial agent prepared by loading Acinetobacter piezoides onto chitosan biochar CHKBC significantly improved the phosphorus solubilization effect by 89.04% compared with the phosphorus solubilization ability of free Acinetobacter piezoides. Compared with the solid bacterial agent prepared by comparative example 2BC, the phosphorus solubilization ability was improved by 65.60%, indicating that the chitosan biochar of the present invention can significantly promote the phosphorus solubilization ability of Acinetobacter piezoides, and is also significantly better than the immobilized bacterial agent prepared by ordinary biochar.

[0139] The present invention has been described in detail above with reference to specific embodiments and material property analysis. However, the above description is only a preferred embodiment of the present invention and does not limit the present invention. Any equivalent substitutions, modifications or improvements to the technical features described in the foregoing embodiments are included within the scope of the present invention.

Claims

1. The application of a microbial solid inoculant in phosphorus solubilization, characterized in that, The aforementioned microbial solid inoculant is prepared by immobilizing microorganisms onto chitosan-loaded biochar. The method for preparing the chitosan-loaded biochar is as follows: Step 1: After drying the biomass, crush and sieve it to obtain usable biomass, and then pyrolyze it to obtain biochar; Step 2: Prepare a potassium hydroxide solution, add biochar and stir with a magnetic stirrer, let stand, then wash repeatedly with distilled water until the pH no longer changes to ensure that all residual KOH is removed. Finally, dry at 60°C, pulverize and pass through an 80-mesh sieve to obtain potassium hydroxide modified biochar. Step 3: Dissolve chitosan in an acidic solution with a degree of deacetylation of over 75%, and stir magnetically until completely dissolved; Step 4: Mix the potassium hydroxide-modified biochar with the prepared chitosan solution and stir, then ultrasonically mix until homogeneous; add sodium hydroxide solution to adjust the pH to 12, continue ultrasonic mixing until homogeneous, and let stand; after standing, filter, wash, dry, grind, and sieve the obtained product to obtain chitosan-loaded biochar. The biomass is one of rice straw, rice husks, corn stalks, or peanut shells; The preparation method of the microbial solid inoculant is as follows: A single colony of Acinetobacter pitride was picked and added to 100 mL of beef extract peptone liquid medium. The medium was then placed on a shaker at 180 r / min and cultured at 30 °C for 20 h until the logarithmic growth phase was reached. Biochar was added to the beef extract peptone medium at a ratio of 3% and autoclaved. Then, Acinetobacter pitride bacterial solution was added to the beef extract peptone medium at a ratio of 1% (v:v). The mixture was cultured in a constant temperature incubator at 30 °C and 180 r / min for 3 days. After culture, the solid microbial inoculum was obtained by centrifugation. The microorganism in question is Acinetobacter pitera, with accession number CCTCC NO: M 2023778.

2. The application as described in claim 1, characterized in that, The concentration of the potassium hydroxide solution is 0.5~1.5 mol / L.

Images