Biochar-based microbial agent, preparation method thereof and application of biochar-based microbial agent in improvement of acidified tea garden soil

Abstract

The invention discloses a biochar-based microbial agent, a preparation method thereof and application of the biochar-based microbial agent in improvement of acidified tea garden soil, belongs to the technical field of microbial agents, and provides a biochar-based microbial agent which is prepared from Serratia mar cescens JW-CZ2 and mushroom dreg biochar. The preparation method of the charcoal-based microbial agent comprises the following steps: drying, crushing and sieving mushroom dreg raw materials to obtain pretreated mushroom dreg powder; carbonizing to obtain mushroom dreg charcoal; mixing with a culture medium to obtain a charcoal culture medium mixture; inoculating serratia marcescens JW-CZ2 into the biochar culture medium mixture, performing shake cultivation, and taking precipitate; and washing and freeze-drying to obtain the biochar-based microbial agent. The biochar-based microbial agent is applied to acidified tea garden soil, the pH of the acidified soil is increased, the soil fertility is improved, and the biochar-based microbial agent is an ideal acidified tea garden soil conditioner.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial agents, and in particular relates to a biochar-based microbial agent, a preparation method thereof, and an application thereof in improving acidified tea garden soil. Background Art

[0002] Tea tree [Camellia sinensis (Linn.) O. Kuntze] is one of the main economic crops, and the area of ​​tea tree cultivation is constantly expanding. Tea trees prefer acid and are afraid of alkali. Long-term cultivation can easily cause soil acidification in tea gardens. However, tea trees are acid-loving crops, and the more acidic the soil, the more suitable it is for tea trees to grow. Studies have shown that the pH range suitable for tea tree growth is 4.5-6.0, among which pH 5.5 is the most suitable for tea tree growth. When the soil pH is lower than 4, the growth of tea trees is inhibited. A large number of studies have shown that soil acidification refers to the amount of H in the soil. + The process of soil pH changing from high to low due to continuous accumulation. The O2 in the soil acidification process mainly comes from the dissociation of carbonic acid, organic acid and water, the addition of physiological acid fertilizers and the precipitation of atmospheric acid substances. Under natural conditions, if the precipitation far exceeds the evaporation, the exchangeable base ions in the soil will move downward with the leaching of rainwater, and the soluble components in the soil will decrease. At this time, the H + Replace the base ions on the soil colloid (i.e. exchangeable K + 、Na + , Ca 2+ Mg 2+ Tea garden soil acidification not only accelerates the loss of soil mineral nutrients, but also aggravates soil compaction and reduces the number and activity of beneficial microorganisms, which can easily lead to the activation of toxic elements such as heavy metals in the soil, affecting the yield and quality of tea, and causing food safety hazards for tea. At the same time, tea garden soil acidification can also lead to the dissolution of soil phosphorus, increase the content of unstable phosphorus in the soil, increase the risk of phosphorus migration and leaching, and aggravate the degradation of soil fertility. Tea garden soil acidification is a major challenge facing the tea industry. Seeking effective soil acidification control technology, improving tea garden soil pH, and maintaining a good tea garden soil environment are of great significance to promoting the tea industry.

[0003] Biochar is a relatively stable solid material with high carbon content obtained by high-temperature pyrolysis and carbonization of organic matter (such as crop straw, wood, etc.) in an oxygen-limited environment. In recent years, the application of biochar in acidified soil improvement has gradually become a research hotspot. Biochar has the advantages of containing alkaline substances and a large amount of essential nutrients and can effectively regulate the cycle of nutrients in the soil. Therefore, it is considered to be an ideal material for improving the acidified soil environment.

[0004] Microbial fertilizer is a new type of fertilizer. It is a kind of fertilizer with microbial life activities as the core. It improves the target crops through microbial activities to obtain beneficial fertilizers. Studies have shown that after inoculating Pseudomonas sp. RU47 into the rhizosphere of tomatoes, the phosphorus absorption of plants increased significantly and promoted plant growth; the combination of biochar and plant growth-promoting rhizobacteria (PGPR) can significantly increase the diversity and enzyme activity of microbial communities in the tomato root soil, increase the nitrogen effectiveness in the soil and the absorption of nitrogen by plants, thereby increasing the accumulation of dry matter. However, free bacteria are easily affected by the external environment. Adverse environmental factors such as high salinity and toxicity can lead to limited growth or even death of microorganisms, making it difficult to achieve bioremediation as expected. It has certain limitations in practical applications, which greatly limits the actual promotion and application. The colonization of microorganisms on suitable carriers can enrich and stabilize microorganisms. Therefore, it is urgent to find a suitable carrier to improve the stability of the effect of microbial use. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes a biochar-based microbial agent, a preparation method thereof and an application in improving acidified tea garden soil. Biochar is prepared by anaerobic pyrolysis of mushroom residue and Serratia marcescens JW-CZ2 is used as raw materials to prepare the biochar-based microbial agent. The mushroom residue biochar has a large pH, specific surface area and rich functional groups, which can provide adsorption sites for Serratia marcescens JW-CZ2. The biochar-based microbial agent is applied to the acidified tea garden soil, which improves the pH of the acidified soil and increases the soil fertility. The biochar-based microbial agent is an ideal acidified tea garden soil conditioner.

[0006] To achieve the above-mentioned purpose, the present invention provides a biochar-based microbial agent, which is prepared by including Serratia marcescens JW-CZ2 and bacterial residue biochar.

[0007] Preferably, the Serratia marcescens JW-CZ2 was deposited in China Center for Type Culture Collection on March 10, 2015, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M2015095.

[0008] Preferably, the mushroom residue biochar is prepared using mushroom residues as raw materials.

[0009] The present invention also provides a method for preparing the biochar-based microbial agent, comprising the following steps:

[0010] (1) drying the mushroom residue raw material, crushing and screening to obtain pretreated mushroom residue powder;

[0011] (2) isolating the pretreated mushroom residue powder obtained in step (1) from air, carbonizing, grinding and sieving to obtain mushroom residue biochar;

[0012] (3) mixing the residue biochar obtained in step (2) with the culture medium and sterilizing the mixture to obtain a biochar culture medium mixture;

[0013] (4) inoculating Serratia marcescens JW-CZ2 into the biochar culture medium mixture obtained in step (3), culturing on a shaking table, first centrifuging, and taking a precipitate;

[0014] (5) The precipitate obtained in step (4) is washed with physiological saline and freeze-dried to obtain a biochar-based microbial agent.

[0015] Preferably, the mushroom residue raw material in step (1) is mushroom residue; the drying temperature in step (1) is 100-110° C., and the drying time is 12 hours; and the particle size of the crushed and sieved material in step (1) is 90-110 mesh.

[0016] Preferably, the air isolation in step (2) is specifically to wrap the pretreated mushroom residue powder with tin foil; the carbonization temperature in step (2) is 300-700°C, and the carbonization time is 1-3h; the particle size of the ground and sieved in step (2) is 90-110 mesh.

[0017] Preferably, in step (3), the ratio of the mixture of the residue biochar and the culture medium is 1 g: 90-110 mL, and the culture medium is LB liquid culture medium.

[0018] Preferably, the inoculation amount of the Serratia marcescens JW-CZ2 in step (4) is calculated according to the volume ratio of the mixture of Serratia marcescens JW-CZ2 and the biochar culture medium of 0.8 to 1.2:100; the temperature of the shaking culture in step (4) is 26 to 30°C, the rotation speed of the shaking culture is 160 to 200 rpm, and the time of the shaking culture is 22 to 26 h.

[0019] Preferably, the washing times in step (5) are 2 to 4 times, the freeze-drying temperature is -67 to -72°C, and the freeze-drying time is 24 hours.

[0020] The present invention also provides the biochar-based microbial agent or the method for preparing the biochar-based microbial agent and the use of the biochar-based microbial agent prepared in improving acidified tea garden soil.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects:

[0022] The biochar-based microbial agent prepared by the present invention uses mushroom residues as raw materials for preparing biochar. The mushroom residues contain a variety of bacterial proteins, metabolites and minerals, and a large amount of mycelium and carbon sources have adsorption effects. Combined with Serratia marcescens JW-CZ2, the strain has efficient phosphorus solubilization ability and good plant growth promotion mechanism, and also has the potential to develop special microbial fertilizers for tea trees, providing a biochar-based microbial agent for improving the survival rate of bacterial storage, improving acidified soil, and recycling agricultural waste.

[0023] The present invention explores the influence of biochar-based microbial agents on soil fertility, soil exchangeability and soil phosphorus components in acidified tea gardens through indoor soil culture experiments. The biochar-based microbial agents are applied to acidified tea garden soil to increase the pH of acidified soil and increase soil fertility; they can reduce soil exchangeability and increase the total amount of soil base; they can increase soil active phosphorus, active phosphorus in the soil, and increase the stable phosphorus content in the soil, which is beneficial to improving the soil phosphorus pool supply capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is an infrared spectrum of the fungus residue biochar prepared in the present invention, in which JZ500 represents the fungus residue biochar of Example 1, JZ300 represents the fungus residue biochar of Example 2, and JZ700 represents the fungus residue biochar of Example 3;

[0026] Figure 2 The scanning electron microscope images of the fungus residue biochar prepared in the present invention, wherein A is the fungus residue biochar of Example 2, B is the fungus residue biochar of Example 1, and C is the fungus residue biochar of Example 3, and the scale is 2 μm;

[0027] Figure 3 It is a scanning electron microscope image of the biochar-based microbial agent prepared by the present invention, wherein A is the biochar-based microbial agent of Example 2, B is the biochar-based microbial agent of Example 1, and C is the biochar-based microbial agent of Example 3, and the scale is 2 μm;

[0028] Figure 4The effect of the biochar-based microbial agent prepared by the present invention on the soil fertility of the tea garden, wherein A is the effective phosphorus content of the soil, B is the effective nitrogen content of the soil, C is the total phosphorus content of the soil, D is the total nitrogen content of the soil, and E is the total potassium content of the soil. In the figure, CK represents the untreated group, JW-CZ2 represents the microbial agent group, JW-CZ2-JZ300 represents the biochar-based microbial agent treatment group of Example 2, JW-CZ2-JZ500 represents the biochar-based microbial agent treatment group of Example 1, and JW-CZ2-JZ700 represents the biochar-based microbial agent treatment group of Example 3;

[0029] Figure 5 The improvement effect of the biochar-based microbial agent prepared by the present invention on tea garden soil, wherein A is soil pH, B is soil electrical conductivity, C is the total amount of soil exchangeable acid, D is soil exchangeable hydrogen, E is soil exchangeable aluminum, F is soil exchangeable potassium, G is soil exchangeable sodium, H is soil exchangeable calcium, I is soil exchangeable magnesium, and J is soil cation exchange capacity. In the figure, CK represents the untreated group, JW-CZ2 represents the microbial agent group, JW-CZ2-JZ300 represents the biochar-based microbial agent treatment group of Example 2, JW-CZ2-JZ500 represents the biochar-based microbial agent treatment group of Example 1, and JW-CZ2-JZ700 represents the biochar-based microbial agent treatment group of Example 3;

[0030] Figure 6 This is the effect of the biochar-based microbial agent prepared by the present invention on the phosphorus components of tea garden soil, wherein A is H2O-P, B is NaHCO3-Pi, C is NaHCO3-Po, D is NaOH-Pi, E is NaOH-Po, F is HCl-P, and G is closed phosphorus. In the figure, CK represents the untreated group, JW-CZ2 represents the microbial agent group, JW-CZ2-JZ300 represents the biochar-based microbial agent treatment group of Example 2, JW-CZ2-JZ500 represents the biochar-based microbial agent treatment group of Example 1, and JW-CZ2-JZ700 represents the biochar-based microbial agent treatment group of Example 3. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] The Serratia marcescens JW-CZ2 of the present invention was deposited in China Center for Type Culture Collection on March 10, 2015, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M2015095.

[0037] The mushroom residue of the invention is collected from an edible fungus factory in Wanzhi District, Wuhu City, Anhui Province.

[0038] Preparation of the preparation used in the present invention:

[0039] LB liquid medium: 3.0 g beef extract, 10.0 g peptone, 3.0 g NaCl, 1000 mL distilled water, pH 7.2-7.4, sterilized at 121°C for 20 min;

[0040] Preparation of bacterial suspension: a single colony of Serratia marcescens JW-CZ2 was placed in 50 mL of LB liquid culture medium and cultured overnight in an air bath shaker at 28°C and 180 rpm to obtain a bacterial suspension of Serratia marcescens JW-CZ2 for later use;

[0041] Physiological saline: Dissolve 9.0 g NaCl in 1000 mL distilled water, shake well, and sterilize at 121°C for 20 min.

[0042] Example 1

[0043] (1) removing impurities from mushroom residue, air-drying, drying at 105° C. for 12 h until constant weight, and crushing through a 100-mesh sieve to obtain pretreated mushroom residue powder;

[0044] (2) The pretreated mushroom residue powder was wrapped with tin foil to isolate the air, placed in a muffle box furnace, carbonized at 500 °C for 2 h, cooled to room temperature, and ground through a 100-mesh sieve to obtain mushroom residue biochar JZ500;

[0045] (3) The residue biochar was mixed with LB liquid culture medium at a ratio of 1 g:100 mL and sterilized at 121 °C for 20 min to obtain a biochar culture medium mixture;

[0046] (4) Inoculate the bacterial suspension of Serratia marcescens JW-CZ2 into the biochar culture medium mixture, the inoculation amount of Serratia marcescens JW-CZ2 is calculated according to the volume ratio of Serratia marcescens JW-CZ2 to the biochar culture medium mixture of 1:100, culture in an air bath shaker at 28°C and 180rpm for 24h, and centrifuge for the first time at 5000rpm and 25°C for 10min to obtain the precipitate;

[0047] (5) The precipitate was washed with physiological saline, centrifuged (5000 rpm, 25°C, 10 min) once, washed three times, and freeze-dried at -70°C for 24 h to obtain the biochar-based microbial agent JW-CZ2-JZ500.

[0048] Example 2

[0049] (1) removing impurities from mushroom residue, air-drying, drying at 105° C. for 12 h until constant weight, and crushing through a 100-mesh sieve to obtain pretreated mushroom residue powder;

[0050] (2) The pretreated mushroom residue powder was wrapped with tin foil to isolate the air, placed in a muffle box furnace, carbonized at 300 °C for 2 h, cooled to room temperature, and ground through a 100-mesh sieve to obtain mushroom residue biochar JZ300;

[0051] (3) The residue biochar was mixed with LB liquid culture medium at a ratio of 1 g: 100 mL and sterilized at 121 °C for 20 min to obtain a biochar culture medium mixture;

[0052] (4) Inoculate the bacterial suspension of Serratia marcescens JW-CZ2 into the biochar culture medium mixture, the inoculation amount of Serratia marcescens JW-CZ2 is calculated according to the volume ratio of Serratia marcescens JW-CZ2 to the biochar culture medium mixture of 1:100, culture in an air bath shaker at 28°C and 180rpm for 24h, and centrifuge for the first time at 5000rpm and 25°C for 10min to obtain the precipitate;

[0053] (5) The precipitate was washed with physiological saline, centrifuged (5000 rpm, 25°C, 10 min) once, washed three times, and freeze-dried at -70°C for 24 h to obtain the biochar-based microbial agent JW-CZ2-JZ300.

[0054] Example 3

[0055] (1) removing impurities from mushroom residue, air-drying, drying at 105° C. for 12 h until constant weight, and crushing through a 100-mesh sieve to obtain pretreated mushroom residue powder;

[0056] (2) The pretreated mushroom residue powder was wrapped with tin foil to isolate the air, placed in a muffle box furnace, carbonized at 700 °C for 2 h, cooled to room temperature, and ground through a 100-mesh sieve to obtain mushroom residue biochar JZ700;

[0057] (3) The residue biochar was mixed with LB liquid culture medium at a ratio of 1 g: 100 mL and sterilized at 121 °C for 20 min to obtain a biochar culture medium mixture;

[0058] (4) Inoculate the bacterial suspension of Serratia marcescens JW-CZ2 into the biochar culture medium mixture, the inoculation amount of Serratia marcescens JW-CZ2 is calculated according to the volume ratio of Serratia marcescens JW-CZ2 to the biochar culture medium mixture of 1:100, culture in an air bath shaker at 28°C and 180rpm for 24h, and centrifuge for the first time at 5000rpm and 25°C for 10min to obtain the precipitate;

[0059] (5) The precipitate was washed with physiological saline, centrifuged (5000 rpm, 25°C, 10 min) once, washed three times, and freeze-dried at -70°C for 24 h to obtain the biochar-based microbial agent JW-CZ2-JZ700.

[0060] Example 4

[0061] (1) removing impurities from mushroom residue, air-drying, drying at 100° C. for 12 h until constant weight, and crushing through a 110-mesh sieve to obtain pretreated mushroom residue powder;

[0062] (2) Wrapping the pretreated mushroom residue powder with tin foil to isolate the air, placing it in a muffle box furnace, carbonizing it at 500°C for 1 hour, cooling it to room temperature, and grinding it through a 110-mesh sieve to obtain mushroom residue biochar;

[0063] (3) The residue biochar was mixed with LB liquid culture medium at a ratio of 1 g:90 mL and sterilized at 121 °C for 20 min to obtain a biochar culture medium mixture;

[0064] (4) Inoculate the bacterial suspension of Serratia marcescens JW-CZ2 into the biochar culture medium mixture, the inoculation amount of Serratia marcescens JW-CZ2 is calculated according to the volume ratio of Serratia marcescens JW-CZ2 to the biochar culture medium mixture of 0.8:100, culture in an air bath shaker at 26°C and 200 rpm for 26 h, and then centrifuge for 10 min at 5000 rpm and 25°C to obtain a precipitate;

[0065] (5) The precipitate was washed with physiological saline once, centrifuged (5000 rpm, 25°C, 10 min) once, washed three times, and freeze-dried at -70°C for 24 h to obtain a biochar-based microbial agent.

[0066] Example 5

[0067] (1) removing impurities from mushroom residue, air-drying, drying at 110° C. for 12 h until constant weight, and crushing through a 90-mesh sieve to obtain pretreated mushroom residue powder;

[0068] (2) Wrapping the pretreated mushroom residue powder with tin foil to isolate the air, placing it in a muffle box furnace, carbonizing it at 500°C for 3 h, cooling it to room temperature, and grinding it through a 90-mesh sieve to obtain mushroom residue biochar;

[0069] (3) The residue biochar was mixed with LB liquid culture medium at a ratio of 1 g:110 mL and sterilized at 121 °C for 20 min to obtain a biochar culture medium mixture;

[0070] (4) Inoculate the bacterial suspension of Serratia marcescens JW-CZ2 into the biochar culture medium mixture, the inoculation amount of Serratia marcescens JW-CZ2 is calculated according to the volume ratio of Serratia marcescens JW-CZ2 to the biochar culture medium mixture of 1.2:100, culture in an air bath shaker at 30°C and 160rpm for 22h, and centrifuge for the first time at 5000rpm and 25°C for 10min to obtain the precipitate;

[0071] (5) The precipitate was washed with physiological saline, centrifuged (5000 rpm, 25°C, 10 min) once, washed three times, and freeze-dried at -67°C for 24 h to obtain the biochar-based microbial agent JW-CZ2-JZ500.

[0072] Experimental Example 1

[0073] 1.1 Test soil

[0074] The samples were collected at the Magu Mountain Tea Tree Breeding Base in Xuanzhou District, Xuancheng City, Anhui Province (118°56′7.11″E, 30°57′37.78″N). The soil type of the base is yellow-brown soil. The specific soil indicators are shown in Table 1.

[0075] Table 1 Physical and chemical properties of the tested soil

[0076]

[0077] 1.2 Experimental design

[0078] Five treatments were set up: no application of biochar microbial agent (CK), application of microbial agent (JW-CZ2), application of biochar-based microbial agent prepared in Example 2 (JW-CZ2-JZ300), application of biochar-based microbial agent prepared in Example 1 (JW-CZ2-JZ500), and application of biochar-based microbial agent prepared in Example 3 (JW-CZ2-JZ700). The details are as follows: 500mL tissue culture bottles were used for culture, and 200.0g of air-dried soil was placed in each bottle. Only an equal amount of sterile water was added to the CK treatment; the microbial agent group and the three biochar-based microbial agents were each added at 2% (m 炭基菌剂 :m 土 ) was added to the bottle, and each treatment was repeated five times. The soil moisture was kept at 75% of the field water holding capacity and placed in an incubator at 25°C for 60 days. The soil moisture content was kept constant during the incubation period. After the incubation period, the soil was taken out and naturally air-dried, and then sieved through a 2 mm sieve for later use.

[0079] 1.3 Test methods

[0080] 1.3.1 Basic physical and chemical properties and characterization of biochar

[0081] The ratio of the mass of the materials before and after pyrolysis was calculated as the yield of biochar. The prepared fungus residue biochar (JZ300, JZ500 and JZ700) was mixed with 1 g:20 mL of deionized water, shaken for 1.5 h, and allowed to stand for 1 h. The supernatant was measured with pH and EC electrodes. The fungus residue biochar was added to a nitric acid solution with a pH of 4 (m 炭 :m 溶液 =1:5) The pH value of the solution was measured after 24 hours of equilibrium; the mass fraction of biochar ash was determined by dry ashing method; the determination of biochar available phosphorus was based on LY / T 1232-2015 "Determination of forest soil phosphorus"; FTIR was used to determine the surface functional groups of biochar; SEM was used for surface morphology analysis; specific surface area was determined by specific surface area tester; and surface potential was measured by Zeta potential analyzer.

[0082] 1.3.2 Characterization of biochar-based microbial agents

[0083] The morphology of JW-CZ2 on biochar was observed using SEM, and the specific surface area was measured using a specific surface area tester.

[0084] 1.3.3 Determination of soil physical and chemical indicators

[0085] Soil pH and EC: measured by acidometer method (soil-water ratio 1:2.5); total soil exchangeable acid (exchangeable hydrogen, exchangeable aluminum): measured by 1 mol / L KCl-neutralization titration method; soil exchangeable base ions: extracted with 1 mol / L ammonium acetate, and Ca in the extract was measured by atomic absorption spectrophotometer 2+ and Mg 2+ Flame photometer was used to determine the K content in the extract. + and Na + Content; Soil available phosphorus: extracted by 0.03mol / LNH4F-0.025mol / L HCl, determined by molybdenum antimony colorimetric method; Tissen phosphorus fractionation method was used to extract soil phosphorus, and determined by molybdenum antimony colorimetric method.

[0086] 1.4 Data Processing

[0087] The results were evaluated by variance analysis using the program SPSS26.0, and the three replicates of the data obtained from each treatment were statistically analyzed. The significance of the differences was statistically processed by one-way ANOVA analysis, and the p values ​​obtained by the significance test method were summarized and analyzed. When p<0.05, the difference was significant, and when p<0.01, the difference was extremely significant; Pearson was used to analyze the correlation between the various indicators of acidified tea garden soil; SPSS26.0 was used for principal component analysis; and Orgin2024b was used for drawing.

[0088] 2 Results and analysis

[0089] 2.1 Basic properties and characterization analysis of biochar

[0090] 2.1.1 Basic physical and chemical properties of fungus residue biochar

[0091] The basic physical and chemical properties of the residue biochar are shown in Table 2. With the increase of pyrolysis temperature, the yield of the residue biochar gradually decreased, and the pH, conductivity, ash content and acid balance capacity gradually increased. The yield of the residue biochar gradually decreased with the increase of temperature. Among them, JZ300 had the highest yield of 54.05%, and JZ700 had the lowest yield of 34.14%. The pH of the three residue biochars was strongly alkaline as a whole, among which JZ300 had the lowest pH of 9.77 and JZ700 had the highest pH of 11.94. When the pyrolysis temperature increased from 300℃ to 700℃, the conductivity of the three residue biochars increased from 1.52ms / cm to 4.74ms / cm; the ash content increased from 22.05% to 33.78%. The effective phosphorus contents of the three kinds of biochars were 1129.77 mg / kg, 1861.15 mg / kg, and 403.93 mg / kg, respectively. When the pyrolysis temperature was between 300 and 500 °C, the effective phosphorus content of biochar increased with the increase of pyrolysis temperature; when the pyrolysis temperature was between 500 and 700 °C, the effective phosphorus content of biochar decreased. The pH of the equilibrium solution reflects the acid balance ability of biochar, which is an important indicator for improving acidic soil. The pyrolysis temperature has a great influence on the acid neutralization ability of the slag carbon. As shown in Table 2, the pH of the solutions of the three kinds of carbon acid balance after 24 hours were 8.49, 9.83, and 11.18, respectively. The acid balance ability of the slag biochar gradually increased with the increase of pyrolysis temperature, from 8.49 to 11.18.

[0092] Table 2 Basic physical and chemical properties of fungus residue biochar

[0093]

[0094] 2.1.2 BET of fungus residue biochar

[0095] The specific surface area, average mesopore diameter, and total pore volume of the fungus residue biochar are shown in Table 3. It can be seen from the table that the specific surface area and total pore volume of the three biochars increase with the increase of pyrolysis temperature, and the specific surface area BET and pore volume of the three biochars reach the maximum value at the thermal decomposition temperature of 700 °C. The specific surface areas of the three fungus residue biochars are 7.76 m 2 / g, 8.99m 2 / g, 37.32m 2 / g, and the total pore volume is 0.022cm 3 / g, 0.026cm 3 / g, 0.064cm 3 / g. The largest change was that compared with JZ300 and JZ500, the specific surface area of ​​JZ700 increased by 380.09% and 315.13%, respectively, and the total pore volume increased by 190.90% and 146.1%, respectively. The average pore size of the fungus residue biochar showed a trend of increasing first and then decreasing with the increase of temperature, reaching a maximum of 12.71nm at 500℃ and a minimum of 6.89nm at 700℃.

[0096] Table 3 BET test and pore size analysis of fungus residue biochar

[0097] Mushroom residue biochar <![CDATA[Specific surface area / (m 2 / g)]]> Average mesopore diameter / (nm) <![CDATA[Total pore volume / (cm 3 / g)]]> JZ300 7.76 11.10 0.022 JZ500 8.99 12.71 0.026 JZ700 37.32 6.89 0.064

[0098] 2.1.3 FTIR of fungus residue biochar

[0099] Fourier transform infrared spectrum of fungus residue biochar Figure 1 As shown, from Figure 1 It can be observed that JZ300, JZ500 and JZ700 all have relatively rich functional groups, and the characteristic absorption peaks are basically the same, indicating that with the increase of pyrolysis temperature, the types of functional groups have not changed significantly, but high-temperature biochar (700℃) shows stronger aromaticity than low-temperature biochar. Functional group area: With the increase of pyrolysis temperature, at 3610cm -1 The -OH absorption peak at 2353 cm-1 gradually weakens. Double bond stretching vibration region: With the increase of pyrolysis temperature, the -OH absorption peak at 2353 cm-1 gradually weakens. -1 The intensity of the organic pH absorption peak at 1540-1646 cm -1 The absorption peaks between 1640cm and 1640cm are generated by the stretching vibration of C=O of carboxyl, carbonyl, ester group and C=C skeleton of aromatic ring. -1 The absorption peak at wavelength is mainly derived from the stretching vibration of C=O bond. Fingerprint region: at 1151cm -1 ~1060cm -1 The absorption peak at 877 cm is attributed to the stretching vibration of organic CO in the biochar structure; -1 ~588cm -1 The absorption peak of the aromaticity = CH out-of-plane bending vibration gradually increases with the increase of pyrolysis temperature, and the aromaticity increases. The higher the aromaticity of the biochar, the more favorable it is to be used as a carrier for immobilized microorganisms.

[0100] 2.1.4 Zeta potential of fungus residue biochar

[0101] Zeta potential is an important indicator to characterize the stability of the dispersed system. The Zeta potentials of the three biochars are shown in Table 4. As shown in Table 4, the Zeta potentials of the three biochars range from -20.8 to -26.7 mV, and their surfaces are negatively charged. With the increase of pyrolysis temperature, they show a trend of first decreasing and then increasing. Among them, JZ300 has the lowest Zeta potential, which is -26.7 mV; JZ500 has the highest Zeta potential, which is -20.8 mV.

[0102] Table 4 Zeta potential analysis of fungus residue biochar

[0103] Mushroom residue biochar Zeta potential (mV) JZ300 -26.7 JZ500 -20.8 JZ700 -24.4

[0104] 2.1.5 SEM of fungus residue biochar

[0105] Scanning electron microscopy (SEM) can quickly characterize the microstructure of prepared biochar materials and provide an important reference for the evaluation of the physical adsorption performance of biochar. Figure 2 The surface morphology of the biochar at different pyrolysis temperatures is shown in Figure 2 Middle A, Figure 2 Medium B and Figure 2 In Figure C, it can be seen that the fungus slag charcoals prepared under different temperature conditions all show obvious pore structure characteristics, but the number and size of pores at different temperatures are different. Especially at 300°C, JZ300 shows a clear pore structure, but compared with other temperature conditions, the number of pores is small and relatively small; when the temperature rises to 500°C, the surface of JZ500 is smoother and the pores become larger, and the specific surface area also increases accordingly; when the temperature reaches 700°C, the surface of JZ700 becomes rough, forming a certain amount of flocculent structure.

[0106] 2.2 Characterization and analysis of biochar-based microbial agents

[0107] 2.2.1 BET of biochar-based microbial agents

[0108] The BET test and pore size analysis of biochar-based microbial agents are shown in Table 5. The specific surface area, average mesopore diameter and total pore volume of the three biochar-based microbial agents increase with the increase of pyrolysis temperature. Among the three biochar-based microbial agents, JW-CZ2-JZ700 has the largest specific surface area, average mesopore diameter and total pore volume, which are 8.88 m 2 / g, 14.28nm, 0.032cm 3 / g. Compared with JZ-CZ2-JZ300 and JW-CZ2-JZ500, the specific surface area of ​​JW-CZ2-JZ700 increased by 30.78% and 12.41%, the average mesopore diameter increased by 10.53% and 7.37%, and the total pore volume increased by 45.46% and 23.08%, respectively.

[0109] Table 5 BET test and pore size analysis of biochar-based microbial agents

[0110] Mushroom residue biochar <![CDATA[Specific surface area / (m 2 / g)]]> Average mesopore diameter / (nm) <![CDATA[Total pore volume / (cm 3 / g) <!-- 8 -->]]> JW-CZ2-JZ300 6.79 12.92 0.022 JW-CZ2-JZ500 7.90 13.30 0.026 JW-CZ2-JZ700 8.88 14.28 0.032

[0111] 2.2.2 SEM of biochar-based microbial inoculants

[0112] Scanning electron microscopy images of different biochar-based microbial agents Figure 3 The surface morphology of JW-CZ2-JZ300, JW-CZ2-JZ500 and JW-CZ2-JZ700 is shown in Figure 3 Middle A, Figure 3 Medium B and Figure 3 In Figure C, a large number of target strains can be seen attached to the surface and pores of the three types of fungus residue biochar. The JW-CZ2 strain attached to the surface of the three types of fungus residue biochar, and the bacterial appearance was oblate. A large number of JW-CZ2 strains adhered to the uneven surface of the biochar through secretions to form a microbial film. It can be considered that JW-CZ2 was successfully inoculated on the biochar and proliferated on the surface and pore structure, indicating that microorganisms can indeed attach to the surface of biochar and the growth environment conditions are good.

[0113] 2.3 Improvement of acidified tea garden soil by biochar-based microbial agents

[0114] 2.3.1 Effects of biochar-based microbial agents on nutrients in acidified tea garden soil

[0115] Effects of different biochar-based microbial agents on nutrients in acidified tea garden soils Figure 4 Compared with CK, adding biochar-based microbial agents can improve the nutrients of acidified tea garden soil, while adding JW-CZ2 has no significant change. Figure 4 As shown in A, compared with CK, adding biochar-based microbial agents can increase the available phosphorus content in the soil. The order of available phosphorus in the soil under different treatments is JW-CZ2-JZ700>JW-CZ2-JZ300>JW-CZ2>JW-CZ2-JZ500>CK, among which JW-CZ2-JZ700 significantly increased by 38.16 mg / kg compared with CK (p<0.05); JW-CZ2, JW-CZ2-JZ300, and JW-CZ2-JZ500 treatments increased by 3.50 mg / kg, 0.39 mg / kg, and 0.68 mg / kg respectively compared with CK, but there was no significant difference. Figure 4 As shown in Figure B, compared with CK, the addition of biochar-based microbial agents can significantly increase the available nitrogen content in the soil. JW-CZ2-JZ300, JW-CZ2-JZ500, and JW-CZ2-JZ700 significantly increased the available nitrogen content by 200.20 mg / kg, 204.04 mg / kg, and 155.32 mg / kg, respectively, compared with CK (p<0.05). Figure 4 Middle C, Figure 4 Medium D and Figure 4 From E, we can see that adding biochar-based microbial agents can increase the content of soil total phosphorus and soil total nitrogen, but has no significant effect on soil total potassium. Figure 4 From C, we can see that compared with CK, total phosphorus in JW-CZ2-JZ300, JW-CZ2-JZ500 and JW-CZ2-JZ700 increased significantly by 217.13mg / kg, 314.67mg / kg and 386.83mg / kg respectively (p<0.05). Figure 4 As shown in Figure D, compared with CK, total nitrogen in JW-CZ2-JZ300 and JW-CZ2-JZ500 increased significantly by 81.57 mg / kg and 80.12 mg / kg respectively (p<0.05), JW-CZ2-JZ700 increased by 19.30 mg / kg, and JW-CZ2 decreased by 73.46 mg / kg. Figure 4 As can be seen from Figure E, the total potassium in the soil under each treatment is in the order of CK>JW-CZ2-JZ300>JW-CZ2-JZ700>JW-CZ2>JW-CZ2-JZ500; compared with CK, there was no significant difference in the total potassium in the acidified tea garden soil under the three biochar-based microbial agents treatments.

[0116] 2.3.2 Effects of biochar-based microbial inoculants on soil exchange performance in acidified tea gardens

[0117] Effects of different treatments on the improvement of acidified tea garden soil Figure 5 Compared with CK, different biochar-based microbial agents can significantly increase soil pH, soil conductivity, soil exchangeable potassium, soil exchangeable sodium, soil exchangeable calcium, soil exchangeable magnesium, and total soil cations, and significantly reduce the total soil exchangeable acid, soil exchangeable hydrogen, and soil exchangeable aluminum. There is no significant difference in the treatment of adding JW-CZ2. Figure 5As shown in Figure A, compared with CK, adding biochar can significantly increase soil pH. The order of soil pH under different treatments is JW-CZ2-JZ500>JW-CZ2-JZ300>JW-CZ2-JZ700>JW-CZ2>CK, among which JW-CZ2-JZ300, JW-CZ2-JZ500, and JW-CZ2-JZ700 significantly increased by 1.73, 1.79, and 1.40 units respectively compared with CK (p<0.05); JW-CZ2 increased by 0.11 units, but there was no significant difference. Figure 5 As shown in Figure B, compared with CK, adding biochar can significantly improve soil electrical conductivity. The order of soil electrical conductivity under different treatments is JW-CZ2-JZ700>JW-CZ2-JZ500>JW-CZ2-JZ300>CK>JW-CZ2, among which JW-CZ2-JZ300, JW-CZ2-JZ500, and JW-CZ2-JZ700 significantly increased by 148.58μS / cm, 163.58μS / cm, and 201.38μS / cm respectively compared with CK (p<0.05). Figure 5 From C, we can see that compared with CK, adding biochar-based microbial agents can significantly reduce the total amount of soil exchangeable acid. The total amount of soil exchangeable acid under different treatments is in the following order: CK>JW-CZ2>JW-CZ2-JZ300>JW-CZ2-JZ500>JW-CZ2-JZ700, among which JW-CZ2-JZ300, JW-CZ2-JZ500, and JW-CZ2-JZ700 significantly decreased by 3.16 cmol / kg, 3.34 cmol / kg, and 3.41 cmol / kg, respectively (p<0.05). Figure 5 As shown in Figure D, compared with CK, the addition of biochar-based microbial agents can significantly reduce soil exchangeable hydrogen. The JW-CZ2-JZ300, JW-CZ2-JZ500, and JW-CZ2-JZ700 treatments were significantly reduced by 0.40 cmol / kg, 0.44 cmol / kg, and 0.48 cmol / kg, respectively, compared with CK (p<0.05), but there was no significant difference among the treatments. Effects of different treatments on soil exchangeable aluminum Figure 5As shown in Figure E, compared with CK, the addition of biochar-based microbial agents can significantly reduce soil exchangeable aluminum. The order of soil exchangeable aluminum under different treatments is CK>JW-CZ2>JW-CZ2-JZ300>JW-CZ2-JZ500>JW-CZ2-JZ700, among which JW-CZ2-JZ300, JW-CZ2-JZ500, and JW-CZ2-JZ700 treatments were significantly reduced by 2.77 cmol / kg, 2.90 cmol / kg, and 2.93 cmol / kg, respectively, compared with CK (p<0.05), but there was no significant difference among the treatments. Effects of different treatments on soil exchangeable potassium Figure 5 As shown in Figure F, compared with CK, the addition of biochar-based microbial agents can increase soil exchangeable potassium. Among them, the JW-CZ2-JZ300, JW-CZ2-JZ500, and JW-CZ2-JZ700 treatments significantly reduced 0.07 cmol / kg, 0.12 cmol / kg, and 0.09 cmol / kg, respectively, compared with CK (p<0.05). The effects of different treatments on soil exchangeable sodium are shown in Figure F. Figure 5 As shown in G, compared with CK, the addition of biochar-based microbial agents can increase soil exchangeable sodium. The soil exchangeable sodium under different treatments is in the order of JW-CZ2-JZ500>JW-CZ2-JZ700>JW-CZ2-JZ300>CK>JW-CZ2. The effects of different treatments on soil exchangeable calcium in tea gardens are shown in Figure 2. Figure 5 As shown in Figure H, compared with CK, the addition of biochar-based microbial agents can increase soil exchangeable calcium. JW-CZ2-JZ300, JW-CZ2-JZ500, and JW-CZ2-JZ700 treatments significantly increased 6.47 cmol / kg, 7.37 cmol / kg, 6.29 cmol / kg, 5.03 cmol / kg, 4.48 cmol / kg, and 4.66 cmol / kg, respectively, compared with CK (p<0.05). The effects of different treatments on soil exchangeable magnesium in tea gardens are shown in Figure H. Figure 5 As shown in Figure 1, compared with CK, the addition of biochar and biochar-based microbial agents can increase soil exchangeable magnesium. Among them, JW-CZ2-JZ300, JW-CZ2-JZ500, and JW-CZ2-JZ700 treatments significantly increased 0.41 cmol / kg, 0.37 cmol / kg, and 0.35 cmol / kg, respectively, compared with CK (p<0.05). The effects of different treatments on the cation exchange capacity of tea garden soil are shown in Figure 1. Figure 5 As shown in J, Figure 5It can be seen from the data that compared with CK, the addition of biochar-based microbial agents can increase the soil cation exchange capacity. The soil cation exchange capacity under different treatments is in the order of JW-CZ2-JZ300>JW-CZ2-JZ500>JW-CZ2-JZ700>JW-CZ2>CK, among which JW-CZ2-JZ300, JW-CZ2-JZ500, and JW-CZ2-JZ700 treatments were significantly increased by 2.51 cmol / kg, 2.17 cmol / kg, and 2.13 cmol / kg respectively compared with CK (p<0.05).

[0118] 2.3.3 Effects of biochar-based microbial agents on phosphorus components in acidified tea garden soils

[0119] (1) Effects of biochar-based microbial agents on active phosphorus in acidified tea garden soils

[0120] Depend on Figure 6 Middle A, Figure 6 Medium B and Figure 6 It can be seen from C that compared with CK, the addition of biochar-based microbial agents in acidified tea garden soil significantly increased the soil active phosphorus content, while the addition of JW-CZ2 strain had no significant effect on soil active phosphorus.

[0121] Depend on Figure 6 As shown in Figure A, compared with CK, different treatments increased soil H2O-P, among which JW-CZ2-JZ500 and JW-CZ2-JZ700 treatments significantly increased the H2O-P content, and the highest H2O-P in JW-CZ2-JZ500 treatment was 40.76 mg / kg.

[0122] Depend on Figure 6 As shown in Figure B, compared with CK, different treatments significantly increased the NaHCO3-Pi content. The JW-CZ2, JW-CZ2-JZ300, and JW-CZ2-JZ500 treatments increased by 40.55 mg / kg, 20.56 mg / kg, and 34.52 mg / kg, respectively (p<0.05); there was no significant difference between JW-CZ2 and JW-CZ2-JZ700 and CK.

[0123] Depend on Figure 6 It can be seen from Figure 3 that compared with CK, the JW-CZ2-JZ300 and JW-CZ2-JZ700 treatments increased the NaHCO3-Po content in the soil, and there was no significant difference between the JW-CZ2 and JW-CZ2-JZ500 treatments and CK, and the content was in the order of JW-CZ2-JZ300>JW-CZ2-JZ700>CK>JW-CZ2-JZ500>JW-CZ2 from high to low.

[0124] (2) Effects of different treatments on medium active phosphorus

[0125] Depend on Figure 6 Medium D and Figure 6 As shown in Figure E, compared with CK, different treatments increased the contents of soil NaOH-Pi and NaOH-Po.

[0126] Depend on Figure 6 As shown in Figure D, compared with CK, different treatments significantly increased the content of soil NaOH-Pi, among which JW-CZ2-JZ300, JW-CZ2-JZ500, and JW-CZ2-JZ700 increased by 123.16 mg / kg, 100.16 mg / kg, and 56.48 mg / kg respectively compared with CK (p<0.05). Among them, the JW-CZ2-JZ300 treatment had a better effect on increasing the content of soil NaOH-Pi.

[0127] Depend on Figure 6 As shown in Figure E, compared with CK, different treatments increased the content of soil NaOH-Po, and the content was JW-CZ2-JZ300>JW-CZ2>JW-CZ2-JZ700>JW-CZ2-JZ500>CK, and there was no significant difference among the treatments.

[0128] (3) Effects of different treatments on low-activity phosphorus

[0129] Depend on Figure 6 Medium F and Figure 6 It can be seen from Figure 5 that compared with CK, the addition of biochar-based microbial agents significantly increased the soil HCl-P and Residual contents, while the addition of JW-CZ2 strain had no significant difference.

[0130] Depend on Figure 6 It can be seen from Figure F that compared with CK, the addition of biochar-based microbial agents increased the soil HCl-P content (p<0.05). The soil HCl-P content under different treatments was JW-CZ2-JZ700>JW-CZ2-JZ500>JW-CZ2-JZ300>JW-CZ2>CK.

[0131] Depend on Figure 6 It can be seen from Figure 5 that compared with CK, biochar-based microbial agents significantly increased the soil Residual content, and JW-CZ2-JZ300, JW-CZ2-JZ500, and JW-CZ2-JZ700 significantly increased the Residual content by 33.65 mg / kg, 42.29 mg / kg, and 81.01 mg / kg respectively compared with CK.

[0132] 2.4 Evaluation of the comprehensive improvement effect of different biochar-based microbial agents on acidified tea garden soil

[0133] 2.4.1 Correlation analysis of various indicators of acidified tea garden soil under different biochar-based microbial agents treatments

[0134] In order to compare the correlation between the various indicators of acidified tea garden soil, correlation analysis was performed on the indicators such as soil pH, electrical conductivity, soil available phosphorus, total exchangeable acidity, and soil phosphorus components of the four different treatments. The correlation results are shown in Tables 6 and 7.

[0135] As shown in Tables 6 and 7, soil pH is significantly correlated with soil available phosphorus and soil total nitrogen (p<0.05); soil pH is extremely significantly correlated with soil conductivity, soil phosphorus, soil available nitrogen, soil exchangeable acid, soil exchangeable hydrogen, soil exchangeable aluminum, soil exchangeable potassium, soil exchangeable sodium, soil exchangeable calcium, soil exchangeable magnesium, soil cation exchange capacity, soil H2O-P, soil NaHCO3-Pi, soil NaOH-Pi, soil HCl-P, and soil Residual (p<0.01), among which pH is negatively significantly correlated with soil exchangeable acid, soil exchangeable hydrogen, and soil exchangeable aluminum. From the correlation analysis results and the previous analysis, it can be seen that although some indicators in the acidified tea garden soil are strongly correlated, due to the limitations of the research conditions and the systematic errors in the research itself, different soil indicators may have different trends and obvious differences among the indicators. In order to avoid the deviation caused by single indicator analysis, the soil indicators of each acidified soil should be comprehensively analyzed to evaluate the comprehensive improvement effect of different treatments on the soil.

[0136] Table 6 Correlation analysis of soil parameters in tea gardens with different acidification treatments Part 1

[0137]

[0138]

[0139] Table 7 Correlation analysis of soil parameters in tea gardens with different acidification treatments Part II

[0140]

[0141]

[0142] Note: * indicates significant correlation (p<0.05); ** indicates extremely significant correlation (p<0.001); X1~X22 represent pH, electrical conductivity, soil available phosphorus, soil total phosphorus, soil available nitrogen, soil total nitrogen, soil total potassium, soil total exchangeable acid, soil exchangeable hydrogen, soil exchangeable aluminum, soil exchangeable potassium, soil exchangeable sodium, soil exchangeable calcium, soil exchangeable magnesium, soil cation exchange capacity, H2O-P, NaHCO3-Pi, NaHCO3-Po, NaOH-Pi, NaOH-Po, HCl-P, and Residual, respectively.

[0143] 2.4.2 Analysis of the effect of principal component analysis and membership function method on the improvement of acidified tea garden soil

[0144] Table 8 Comprehensive improvement evaluation of acidified tea garden soil under different treatments

[0145]

[0146] Considering that the improvement effect of different treatments on acidified tea garden soil is determined by multiple factors and indicators, a single indicator is one-sided and cannot truly reflect the comprehensive improvement effect of different treatments on soil. Therefore, in order to clarify the improvement effect of different biochar-based microbial agents on acidified tea garden soil and avoid the unreality and inaccuracy of a single evaluation indicator, this study used principal component analysis and soil comprehensive evaluation index (SQI) to analyze the improvement effect of different biochar-based microbial agents on acidified tea garden soil.

[0147] The calculation formula of soil comprehensive evaluation index (SQI) is as follows:

[0148]

[0149] Where W i is the weight value of each soil index, F(X i ) is the membership function value of each soil index. The formula and method of the membership value of each soil index in the formula are as follows:

[0150] X (u) =(XX min ) / (X max -X min );

[0151] X (u) =1-[(XX min ) / (X max -X min )];

[0152] Where: X is the measured value of a certain indicator under each treatment, X max and X minare the maximum and minimum values ​​of the index under specific treatment conditions, respectively. (u) is the membership function value of each soil index. If the soil index is positively correlated with the improvement effect, then X (u) =(XX min ) / (X max -X min ) calculation; if the soil index is negatively correlated with the improvement effect, then use X (u) =1-[(XX min ) / (X max -X min )] calculation. Since different soil indicators have different units and different degrees of variation, if the original data is used, the comprehensive evaluation results are inaccurate. Therefore, the evaluation often requires standardization of the original data. This paper first calculates the membership function of each treatment principal component, and then calculates the SQI. The larger the value, the better the comprehensive improvement effect.

[0153] 3 Effect of pyrolysis temperature on the basic properties of fungus residue biochar

[0154] The yield of the three types of slag biochars with different pyrolysis temperatures showed a decreasing trend with the increase of temperature. This phenomenon can be attributed to the fact that at higher pyrolysis temperatures, a large amount of cellulose and hemicellulose in the slag were gradually decomposed, and the organic matter gradually decreased. The pH of the three types of slag biochars gradually increased and became strongly alkaline. This is because the ash of biochar contains inorganic minerals such as calcium, magnesium, potassium, sodium and other alkaline substances, so the three types of biochar are strongly alkaline as a whole. At 700℃, the high-boiling point substances in the raw material components evaporated more and the content of inorganic mineral components increased, resulting in the highest pH value. At the same time, the FTIR graph inferred that the oxygen-containing functional groups on the surface of the biochar decreased with the increase of pyrolysis temperature. As the temperature gradually rises during the pyrolysis process, the acidic volatile substances in the biochar begin to decrease, while the ash content shows an upward trend. Excessive ash content will lead to a decrease in the specific surface area and pore volume of the biochar, thereby affecting its adsorption and storage properties. In addition, the ash contains a large amount of alkaline minerals such as calcium, magnesium, potassium, sodium and carbonates, which lead to an increase in conductivity.

[0155] The effective phosphorus content of the slag biochar was the highest at 500°C and the lowest at 700°C. When the pyrolysis temperature reached 700°C, the content of effective phosphorus in the biochar decreased. This is because the phosphorus in the biomass pyrolysis at a higher temperature will react with Al 3+ , Ca 2+ Mg 2+The results show that the biochar prepared at a lower temperature is more conducive to the improvement of the effectiveness of phosphorus itself. Therefore, the effective phosphorus content of JZ500 is higher than that of JZ700. The acid neutralization capacity of the three kinds of fungus residue biochars gradually increases. This is because when the pyrolysis temperature is lower than 550℃, it is mainly the loss of water and the decomposition of organic matter. The acid neutralization capacity of biomass charcoal prepared in this temperature range is low and does not change much with the pyrolysis temperature. When the pyrolysis temperature is between 550 and 750℃, as the pyrolysis temperature increases, carbonates gradually decompose. The acid neutralization capacity of fungus residue biochar prepared in this temperature range increases significantly and increases significantly with the increase of pyrolysis temperature.

[0156] 4 Characterization of pyrolysis temperature on biochar from fungus residue

[0157] When the pyrolysis temperature rises, most of the cellulose and lignin in the mushroom residue are decomposed and a large amount of volatile gases are released, which leads to an increase in the number of micropores and specific surface area of ​​the biochar, thus improving the adsorption performance. -1 The absorption peak of -OH at 1151cm-1 gradually weakens. This is because during the carbonization process, the biomass undergoes dehydration and dehydroxylation, resulting in a significant reduction in hydroxyl groups and a significantly weaker absorption peak. At high pyrolysis temperatures (≥500°C), the absorption peak of C=O bonds weakens because C=O bonds are more easily pyrolyzed into CO and CO2. -1 ~1060cm -1 The absorption peak at 877 cm is attributed to the organic CO stretching vibration in the biochar structure, indicating that the three types of biochar have a typical carbon-based skeleton structure and certain oxygen-containing functional groups; -1 ~588cm -1 The absorption peak at the position represents aromaticity = CH out-of-plane bending vibration. As the pyrolysis temperature increases, the absorption peak gradually increases, and the aromaticity increases.

[0158] The Zeta potentials of the three types of fungus residue biochars are similar, with JZ500 having the highest Zeta potential. In a neutral or alkaline environment, the surface of bacteria carries a negative charge, and during the process of bacterial adsorption to the carrier surface, it is usually affected by van der Waals forces and electrostatic forces. As the Zeta potential of the carrier surface increases, its electrostatic attraction to negatively charged microorganisms increases accordingly, resulting in a decrease in the electrostatic repulsion between the negatively charged microorganisms and the carrier, which is beneficial to the immobilization of the microorganisms.

[0159] 5 Characterization of biochar-based microbial agents

[0160] A large number of target strains were attached to the surfaces and voids of the three biochar-based microbial agents, indicating that the large specific surface area and porous structure of the three types of fungus residue biochars can provide a large attachment space for microorganisms and promote the growth and reproduction of strains. When biochar is used as a carrier for microorganisms and put into the soil, the bacteria will be stored in the biochar in advance, which can avoid being affected by environmental factors, thereby improving the competitiveness of microorganisms with other bacteria in the soil and making it easier for microorganisms to survive in the soil. Through BET and SEM analysis of the three biochar-immobilized microorganisms, it was found that the specific surface area of ​​the three types of fungus residue biochars decreased before and after adsorption, and a large number of microorganisms existed on the surface and voids of the fungus residue biochars. This may be due to the bacterial load, which blocked the internal micropores of the biochar and reduced its specific surface area. The increase in the mesopore diameter after biochar was loaded with microorganisms may be caused by the growth and metabolic activities of microorganisms, which produce gas and increase the porosity of biochar.

[0161] 6. Improving the fertility of acidified tea garden soil by biochar-based microbial agents

[0162] Adding three different biochar microbial agents can improve soil nutrients. When biochar is mixed with soil, the porosity and permeability of the soil will also increase. Soils with low bulk density and high organic matter are more conducive to releasing and retaining nutrients. In addition, biochar can enhance the nitrogen cycle of soil microorganisms and promote the growth of nitrogen-fixing microorganisms. Compared with CK, different biochar-based microbial agents can significantly increase the content of soil available nitrogen and total nitrogen. Phosphorus is an important component of macromolecules in crops, and sufficient phosphorus content in the soil is crucial to increasing crop yields. The application of biochar-based microbial agents increases the content of soil available phosphorus and total phosphorus, which may be because biochar helps to improve the utilization efficiency of P in the soil through local pH buffering reactions, thereby increasing the availability of P to plants. The addition of biochar-based microbial agents to acidic tea garden soil had no significant effect on soil total potassium. The reason is that, on the one hand, biochar-based microbial agents contain more soluble phosphates and salt-based substances such as K, Ca and Mg, which significantly increase the input of soil P, K, Ca and Mg after application; secondly, biochar-based microbial agents significantly increase soil pH after application, thereby reducing the fixation of phosphorus by the soil; thirdly, biochar-based microbial agents retain the microporous structure of biochar, which can provide a good living environment for soil microorganisms, increase the activity of soil phosphate-dissolving bacteria and silicate potassium-dissolving bacteria, and increase the activity of enzymes related to soil potassium conversion, thereby increasing the effective nutrient content of tea garden soil. Therefore, it must be pointed out that after exogenous substances enter the soil, the impact on the soil is long-term and complex, and the impact of biochar-based microbial agents on soil nutrients requires long-term research. In general, the application of biochar-based microbial agents can improve the fertility characteristics of acidified tea garden soil to a certain extent.

[0163] 7 Improvement of soil exchange properties in acidified tea gardens by biochar-based microbial agents

[0164] The application of biochar-based microbial agents can increase soil pH, conductivity, the total amount of soil exchangeable bases (exchangeable potassium, sodium, calcium, magnesium), and reduce soil exchangeable acids (exchangeable hydrogen, aluminum). Soil pH is a basic property of soil, which can indirectly reflect the fertility of the soil and its water holding capacity. The application of biochar-based microbial agents increases soil pH compared to CK, mainly because the test biochar contains more alkaline groups and base ions, which can reduce the exchangeable hydrogen content in the soil. When biochar-based microbial agents are applied to acidified tea garden soil, they can not only neutralize the H in the soil, but also reduce the exchangeable hydrogen content in the soil. + and H released by tea tree roots + , and can increase the soil cation exchange capacity and basic substance content. The treatments with the addition of biochar-based microbial agents increased the soil pH, but the soil pH of each treatment was still weakly acidic, which is suitable for the growth of tea trees. Exchangeable acid is one of the capacity indicators of soil acidity. Biochar-based microbial agents effectively reduced the exchangeable acid of the soil. There may be three reasons for this result: First, the application of biochar increases the pH of the soil. When the soil pH>5, aluminum ions hydrolyze to form hydroxyaluminum or aluminum hydroxide precipitation, thereby reducing the exchangeable acid of the soil. Second, the functional groups on the surface of biochar will combine with aluminum to form stable complexes or chelates, thereby reducing the content of exchangeable acid in the soil. Third, the basic ions contained in biochar will react with exchangeable aluminum to reduce the content of exchangeable acid. Soil cation exchange capacity (CEC) is an important indicator for evaluating adsorption performance. The higher the CEC, the stronger the ability to adsorb cations. The application of biochar-based microbial agents can increase CEC. This may be because the cations contained in the biochar-based microbial agents will enhance the ionic strength of the solution and the negative charge density of the soil, thereby increasing the soil CEC. The total amount of soil exchangeable bases includes soil exchangeable potassium, sodium, calcium, and magnesium. The application of biochar-based microbial agents can increase the total amount of exchangeable bases. This may be because biochar reduces the exchangeable aluminum in the soil and increases the surface load, which is K + 、Na + , Ca 2+ Mg 2+ It provides a large number of adsorption sites, increases the adsorption capacity of soil cations, and further increases the total amount of soil exchangeable bases.

[0165] 8 Improvement of phosphorus components in acidified tea garden soil by biochar-based microbial agents

[0166] The application of biochar-based microbial agents can transform soil phosphorus components, activate soil phosphorus, and improve soil fertility. Soil H2O-P and NaHCO3-Pi are active phosphorus, which are easily absorbed and utilized by plants. In the case of extreme phosphorus deficiency, H2O-P is regarded as a direct supplementary source of soil available phosphorus. Therefore, increasing the content of soil H2O-P and NaHCO3-Pi is of great significance to improving soil fertility and promoting plant growth. The application of biochar-based microbial agents in acidified tea garden soil can increase the content of soil H2O-P and NaHCO3-Pi. NaHCO3-Po exists in the form of microbial biomass phosphorus, which is easy to mineralize and can be absorbed and utilized by crops in a short period of time. The application of biochar-based microbial agents increased the content of NaHCO3-Po compared with CK, but there was no significant difference. Since phosphorus in the cells is returned to the soil during the decomposition of soil microbial residues, microbial biomass phosphorus is considered to be the main source of NaHCO3-Po. In addition, microbial biomass phosphorus has also been shown to supplement NaHCO3-Pi. NaOH-Pi and NaOH-Po are potential sources of phosphorus in the soil. They are easy to combine with iron and aluminum oxides in the soil to form insoluble phosphates, which require long-term geochemistry to be absorbed and utilized by plants. It was found that the application of biochar-based microbial agents can increase the content of NaOH-Pi and NaOH-Po in the soil. Because soil organic matter robs the adsorption sites of phosphorus on the surface of iron and aluminum oxides, or forms a thin film on the surface of iron and aluminum oxides, it reduces the adsorption capacity of iron and aluminum oxides on soil phosphorus and increases the content of NaOH-Po and NaOH-Pi in the soil. In addition, since the red soil in the tea garden is generally acidic, aluminum is enriched in large quantities, and aluminum oxides are easy to combine with NaOH-Pi and NaOH-Po in the soil to form insoluble phosphates, thereby indirectly increasing the content of NaOH-Po and NaOH-Pi and increasing the potential source of phosphorus in the soil. Soil HCl-P and Residual are stable sources of phosphorus, which are difficult to be absorbed and utilized by plants. The application of biochar-based microbial agents can increase the content of HCl-P and Residual in the soil.

[0167] 9 Conclusion

[0168] (1) With the increase of pyrolysis temperature, the residue biochar prepared under different pyrolysis temperatures has a large specific surface area and rich pore structure, and the surface contains rich functional groups; a large number of target strains are loaded on the surface of different residue biochar-based microbial inoculants, indicating that the microorganisms have successfully attached to the biochar, and the surface properties and internal pore structure of the biochar provide ideal environmental conditions for the settlement and reproduction of Serratia marcescens JW-CZ2.

[0169] (2) The application of different biochar-based microbial agents to acidified tea garden soil can increase the pH of acidified soil and increase soil fertility; it can reduce soil exchangeable acid and increase the total amount of soil base; it can increase soil active phosphorus, active phosphorus in the soil, and increase the stable phosphorus content in the soil, which is beneficial to improving the soil phosphorus pool supply capacity.

[0170] (3) Through comprehensive analysis, JW-CZ2-JZ500 has a good effect on improving acidified tea garden soil and is a reliable method to improve the survival rate of bacterial storage, improve acidified soil, and reuse agricultural waste.

[0171] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A biochar-based microbial agent, characterized in that: The biochar is prepared from Serratia marcescens JW-CZ2 and bacterial residue biochar.

2. The biochar-based microbial agent according to claim 1, characterized in that: The Serratia marcescens JW-CZ2 was deposited in China Center for Type Culture Collection on March 10, 2015, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M2015095.

3. The biochar-based microbial agent according to claim 1, characterized in that: The mushroom residue biochar is prepared by using mushroom residue as raw material.

4. The method for preparing the biochar-based microbial agent according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) drying the mushroom residue raw material, crushing and screening to obtain pretreated mushroom residue powder; (2) isolating the pretreated mushroom residue powder obtained in step (1) from air, carbonizing, grinding and sieving to obtain mushroom residue biochar; (3) mixing the residue biochar obtained in step (2) with the culture medium and sterilizing the mixture to obtain a biochar culture medium mixture; (4) inoculating Serratia marcescens JW-CZ2 into the biochar culture medium mixture obtained in step (3), culturing on a shaking table, first centrifuging, and collecting a precipitate; (5) The precipitate obtained in step (4) is washed with physiological saline and freeze-dried to obtain a biochar-based microbial agent.

5. The preparation method according to claim 4, characterized in that: The mushroom residue raw material in step (1) is mushroom residue; the drying temperature in step (1) is 100-110° C., and the drying time is 12 hours; the particle size of the crushed and sieved material in step (1) is 90-110 meshes.

6. The preparation method according to claim 4, characterized in that: The isolation from air in step (2) is specifically to wrap the pretreated mushroom residue powder with tin foil; the carbonization temperature in step (2) is 300-700° C., and the carbonization time is 1-3 hours; the particle size of the ground and sieved in step (2) is 90-110 mesh.

7. The preparation method according to claim 4, characterized in that: In step (3), the ratio of the mixture of the residue biochar and the culture medium is 1 g: 90-110 mL, and the culture medium is LB liquid culture medium.

8. The preparation method according to claim 4, characterized in that: The inoculation amount of the Serratia marcescens JW-CZ2 in step (4) is calculated according to the volume ratio of the mixture of Serratia marcescens JW-CZ2 and the biochar culture medium of 0.8-1.2:100; the temperature of the shaking table culture in step (4) is 26-30°C, the rotation speed of the shaking table culture is 160-200rpm, and the time of the shaking table culture is 22-26h.

9. The preparation method according to claim 4, characterized in that: The washing times in step (5) are 2 to 4 times, the freeze-drying temperature is -67 to -72°C, and the freeze-drying time is 24 hours.

10. Use of the biochar-based microbial agent prepared by the preparation method of the biochar-based microbial agent according to any one of claims 1 to 3 or the biochar-based microbial agent according to any one of claims 4 to 9 in improving acidified tea garden soil.

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