Bacillus licheniformis, fermentation inoculant containing bacillus licheniformis, preparation method of fermentation inoculant, organic fertilizer, preparation method of organic fertilizer and planting method of crops

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus licheniformis, a fermentation agent containing the bacillus licheniformis, a preparation method of the fermentation agent, organic fertilizer, a preparation method of the organic fertilizer and a planting method of crops. The preservation number of the bacillus licheniformis is GDMCC No: 66520. The strain is only added as a zymophyte to compost the agaricus bisporus residues, so that the quick decomposition of the agaricus bisporus residues can be realized. And the decomposed product has a good promotion effect on crops, especially tomatoes.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to Bacillus licheniformis, fermentation agents containing it and their preparation methods, organic fertilizers and their preparation methods, and methods for planting crops. Background Technology

[0002] The continuous development of the edible fungi industry has led to the generation of a large amount of fungi residue, putting enormous pressure on environmental protection. Research on the resource utilization of fungi residue mainly focuses on secondary cultivation of edible fungi, organic fertilizer production, soilless cultivation substrate preparation, bioremediation, biogas production, and feed production. Wang Mengmeng et al. found that adding cow manure to mushroom residue during fermentation resulted in rapid temperature rise in the compost pile, a prolonged high-temperature period, and improved compost quality. Meng Xianghai et al.'s research indicates that simplified treatment of fungi residue helps preserve nutrients while meeting harmless standards and facilitating its return to the field. With the deepening research on the biodegradation of lignocellulose, more and more cellulose-degrading bacteria are being applied to practical production. Studies have found that single strains produce only a limited range of enzymes, making it difficult to disrupt the stable lignocellulose network structure and achieve efficient degradation of fungi residue. The compound bacterial agent developed by Feng Hongmei et al. demonstrates stronger enzyme production capabilities than its constituent single bacterial agents. Compound bacterial agents composed of 2-6 strains produce higher degradation effects. Adding a certain proportion of cellulose-degrading bacterial agents to fungi residue can improve fermentation efficiency and achieve the goal of forming high-quality compost. A patent document reports a method for producing organic fertilizer, which includes the following steps: (1) mixing Bacillus licheniformis, Bacillus subtilis, Bacillus stearothermophilus, and Trichoderma reesei in a mass ratio of 1:1:3:0.5 to obtain a fermentation agent, wherein Bacillus licheniformis is ATCC 39326, Bacillus subtilis is ATCC 31595, Bacillus stearothermophilus is ATCC 7953, and Trichoderma reesei is ATCC 39326. 56764; (2) The fermentation agent obtained in step (1) is mixed with cassava residue, rice bran, edible fungus residue and beer lees in a mass ratio of (0.02~0.05):(1~1.5):(15~20):(20~30):(0.3~0.5) to obtain fermentation raw materials; (3) The fermentation raw materials in step (2) are piled into a long fermentation stack and fermented to prepare organic fertilizer; The edible fungus residue is one or a mixture of several of the following: oyster mushroom residue, shiitake mushroom residue and enoki mushroom residue. Patent literature also reports a bio-organic fertilizer made from edible fungi residue suitable for grain crops and fruit trees, mainly composed of the following raw materials by weight percentage: 75-80% edible fungi residue, 8-10% urea, 5-8% monoammonium phosphate, 4-6% potassium sulfate, 1-1.5% microbial bacteria, and 0.2-0.3% regulator; the regulator is a mixture of tetramethylglutaric acid, DA-6 and sodium 5-nitroguaiacol; the weight ratio of tetramethylglutaric acid, DA-6 and sodium 5-nitroguaiacol is 1:1-3:1-2; the microbial bacteria are in powder form, a technology transfer product of the Henan Academy of Sciences, registration certificate number 9412008Y1431, which mainly contains *Azotobacter brasiliensis*, *EM* bacteria and *Bacillus licheniformis*.

[0003] Although there are reports on using microorganisms to ferment mushroom residue for resource reuse, current fermentation strains, especially single strains, are difficult to use for fermenting *Agaricus bisporus* mushroom residue to achieve its reuse. Therefore, this application is submitted. Summary of the Invention

[0004] Based on this, one or more embodiments of this application provide Bacillus licheniformis, fermentation agents containing it, methods for preparing the same, organic fertilizers, methods for preparing the same, and methods for planting crops. These include the following technical solutions:

[0005] One or more embodiments of this application provide a Bacillus licheniformis X3, with accession number GDMCC No:66520.

[0006] One or more embodiments of this application provide a fermentation agent, the fermentation agent comprising Bacillus licheniformis X3.

[0007] One or more embodiments of this application provide a method for preparing a fermentation agent, the preparation method comprising the following steps: inoculating the aforementioned Bacillus licheniformis X3 into a fermentation medium, culturing, and preparing the agent.

[0008] In some embodiments of this application, the cultivation satisfies one or more of the following conditions:

[0009] (1) The fermentation medium includes CMC-Na liquid medium;

[0010] (2) The temperature is 45℃~55℃;

[0011] (3) Rotation speed is 170 r / min -1 ~190 r min -1 ;as well as,

[0012] (4) Culture until Bacillus licheniformis X3 is in the logarithmic phase.

[0013] In some embodiments of this application, Bacillus licheniformis X3 in the logarithmic phase is used for inoculation.

[0014] One or more embodiments of this application provide a method for preparing organic fertilizer, the method comprising the following steps:

[0015] Organic fertilizer is prepared by fermenting the residue of *Bacillus licheniformis* using the aforementioned Bacillus licheniformis X3, the aforementioned fermentation agent, or the fermentation agent prepared by the aforementioned preparation method.

[0016] In some embodiments of this application, the organic fertilizer is prepared by composting.

[0017] In some embodiments of this application, composting satisfies one or more of the following conditions:

[0018] (1) The ratio of the amount of the fermentation agent to the amount of mushroom residue is (8~12) mL: (1800~2200) g;

[0019] (2) The initial C / N ratio of the composting system is 20~30;

[0020] (3) The composting time is 7 days to 28 days; and,

[0021] (4) The compost pile is not turned over during the composting process;

[0022] Optionally, urea can be used as a nitrogen source to adjust the C / N ratio of the initial composting system;

[0023] Optionally, the ratio of the fermentation agent, the urea and the mushroom residue is (8~12) mL: (20~28) g: (1800~2200) g.

[0024] One or more embodiments of this application provide an organic fertilizer prepared by the preparation method described above.

[0025] One or more embodiments of this application provide a method for planting crops, wherein the organic fertilizer is used during the crop planting process;

[0026] Optionally, the crop includes vegetables;

[0027] Optionally, the vegetables include one or more of tomatoes and peppers.

[0028] Compared with traditional technologies, this application has the following advantages:

[0029] This application provides a strain of Bacillus licheniformis X3. Adding this strain alone as a fermentation agent to compost button mushroom substrate enables rapid decomposition of the substrate. The decomposed product has a beneficial effect on crops, especially tomatoes. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The standard curve for glucose (A) and the activity of C4 bacterial agent FPA within 72 h (B) are shown.

[0032] Figure 2 The changes in temperature (A) and moisture (B) during the composting process.

[0033] Figure 3 The values ​​represent the changes in pH (A) and EC (B) during the composting process.

[0034] Figure 4 The changes in TOC (A) and Kjeldahl nitrogen (B) during the composting process are shown.

[0035] Figure 5 The changes in C / N (A) and T value (B) during the composting process.

[0036] Figure 6 The changes in GI during the composting process (A) and the heavy metal content at the end of composting (B).

[0037] Figure 7 The changes in plant height (A) and diameter (B) of the three seedlings are shown.

[0038] Figure 8 The changes in total fresh weight (A) and aboveground fresh weight (B) of the three seedlings are shown.

[0039] Figure 9 The changes in root fresh weight (A) and crown width (B) of three seedlings are shown.

[0040] Figure 10 The changes in dry weight (A) and dry matter content (B) of the three seedlings are shown.

[0041] The Bacillus licheniformis X3 strain provided in this application, with the taxonomic name Bacillus licheniformis, was deposited on June 16, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No:66520. This strain was received and registered by the collection center on June 16, 2025, and was confirmed to be a viable strain by the collection center on the same day. Detailed Implementation

[0042] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0044] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0045] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0046] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0047] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0048] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0049] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0050] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0051] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0052] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0053] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0054] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0055] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0056] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0057] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0058] In a first aspect of this application, a Bacillus licheniformis X3 is provided, with accession number GDMCC No:66520.

[0059] A second aspect of this application provides a fermentation agent comprising Bacillus licheniformis X3.

[0060] It is understood that this application does not impose any particular limitation on the dosage form of the microbial agent; it can be a solid dosage form or a liquid dosage form.

[0061] A fourth aspect of this application provides a method for preparing a fermentation agent, the method comprising the following steps: inoculating the aforementioned Bacillus licheniformis X3 into a fermentation medium, culturing, and preparing the agent.

[0062] In some embodiments of this application, the cultivation satisfies one or more of the following conditions:

[0063] (1) The fermentation medium includes CMC-Na liquid medium;

[0064] (2) The temperature is 45℃~55℃, for example, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55℃;

[0065] (3) Rotation speed is 170 r / min -1 ~190 r min -1 For example, 170, 175, 180, 185, 190 r min. -1 ;as well as,

[0066] (4) Culture until Bacillus licheniformis X3 is in the logarithmic phase.

[0067] In some embodiments of this application, Bacillus licheniformis X3 in the logarithmic phase is used for inoculation.

[0068] A fifth aspect of this application provides a method for preparing organic fertilizer, the method comprising the following steps:

[0069] Organic fertilizer is prepared by fermenting the residue of *Bacillus licheniformis* using the aforementioned Bacillus licheniformis X3, the aforementioned fermentation agent, or the fermentation agent prepared by the aforementioned preparation method.

[0070] In some embodiments of this application, the organic fertilizer is prepared by composting.

[0071] In some embodiments of this application, composting satisfies one or more of the following conditions:

[0072] (1) The ratio of the amount of the fermentation agent to the amount of the mushroom residue is (8~12) mL: (1800~2200) g, for example, 8 mL: 1800 g, 8 mL: 2000 g, 8 mL: 2200 g, 10 mL: 1800 g, 10 mL: 2000 g, 10 mL: 2200 g, 12 mL: 1800 g, 12 mL: 2000 g, 12 mL: 2200 g.

[0073] (2) The initial C / N ratio of the composting system is 20~30, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30;

[0074] (3) The composting time is 7 days to 28 days, for example, 7, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 days; and,

[0075] (4) The compost pile is not turned over during the composting process;

[0076] Optionally, urea can be used as a nitrogen source to adjust the C / N ratio of the initial composting system;

[0077] Optionally, the ratio of the fermentation agent, the urea, and the mushroom residue is (8~12) mL:(20~28) g:(1800~2200) g, for example, 8 mL:24 g:1800 g, 8 mL:28 g:2000 g, 8 mL:20 g:2200 g, 10 mL:28 g:1800 g, 10 mL:24 g:2000 g, 10 mL:20 g:2200 g, 12 mL:28 g:1800 g, 12 mL:20 g:2000 g, 13 mL:20 g:2200 g.

[0078] A sixth aspect of the embodiments of this application provides an organic fertilizer prepared by the aforementioned preparation method.

[0079] A seventh aspect of this application provides a method for planting crops, wherein the organic fertilizer is used during the planting process of the crops;

[0080] Optionally, the crop includes vegetables;

[0081] Optionally, the vegetables include one or more of tomatoes and peppers.

[0082] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0083] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0084] This application screens thermophilic strains with strong cellulose degradation capabilities from fresh button mushroom substrate, prepares inoculant agents, and uses these agents to compost the substrate. Pepper, tomato, and chamomile seedlings are the primary research subjects to investigate the effects of the inoculant agents on compost quality. Specifically, the following content is involved:

[0085] 1. Materials and Methods

[0086] 1.1 Experimental Materials and Equipment

[0087] Sodium carboxymethyl cellulose (CMC-Na) medium (Meng Xianghai, Wang Baicheng, Zhang Xingzhe, et al. Identification of microbial diversity in edible fungus residue treated with different methods and its correlation analysis with environmental factors [J]. Chinese Journal of Soil and Fertilizer, 2023, (04): 200-209.), filter paper strip liquid medium (Wang Lingxiao, Chang Ruixue, Wang Jue, et al. Transformation of organic matter during composting and its evaluation method [J]. Journal of China Agricultural University, 2023, 28(09): 167-175.), 1 g L -1 Congo red staining solution, 1 mol L -1 Sodium chloride solution, 0.1 mol / L -1 Citric acid 0.1 mol L -1 Sodium citrate and glucose standard solution (1 g / L) -1 ), 3,5-dinitrosalicylic acid (DNS) reagent, 0.05 mol / L -1 Citric acid-sodium citrate buffer solution (pH 4.8).

[0088] The button mushroom substrate was collected from Lianmei Agricultural Products Co., Ltd. in Funan County and stored at 4℃ for later use; the radish seeds were sourced from Keda Seed Co., Ltd. in Henan Province (germination rate ≥85%).

[0089] The cultivation soil was sourced from farmland surrounding Fuyang Normal University.

[0090] Seedlings of three crops—chili peppers, tomatoes, and chamomile—were purchased from the Fuyang Agricultural Market, and several plants with similar growth were selected.

[0091] Main instruments: PCR instrument (PTC-20, MJ Research), inductively coupled plasma mass spectrometer (ICP-MS7800, Agilent), precision balance (BSA224S, Sartorius), intelligent biochemical incubator (SHP-250, Shanghai Guangdu), electronic thermometer, intelligent light incubator (GAL-P280C, Dascatel).

[0092] 1.2 Research Methods

[0093] 1.2.1 Preparation of thermophilic cellulose-degrading bacterial agent

[0094] (1) Enrichment, isolation and purification of cellulose-degrading bacteria

[0095] Weigh 5 g of button mushroom substrate and place it in an Erlenmeyer flask. Add 100 mL of distilled water and incubate at 50°C and 180 rpm. -1 Prepare a suspension by shaking for 30 min. Pipette 1 mL of the suspension into an Erlenmeyer flask containing 100 mL of CMC-Na liquid culture medium, and incubate at 50°C and 180 rpm. -1 Incubate under the specified conditions for 12 h to prepare an enrichment solution. Take 1 mL of the enrichment solution and serially dilute it to 10⁻⁶. -4 10 -5 10 -6 100 μL of bacterial culture was taken and spread onto CMC-Na solid medium and cultured at 50℃ for 24 h. Strains with different morphologies were picked, streaked, and purified to the 4th or 5th generation.

[0096] (2) Secondary screening of cellulose-degrading bacteria

[0097] The strains obtained from the initial screening were cultured on CMC-Na solid medium at 50°C for 48 h. Using 1 g L... -1 Stain with Congo red for 15 min, using 1 mol L -1 The cells were eluted with NaCl solution, and the ratio of the diameter of the transparent zone to the diameter of the colony (D / d) was calculated to compare the cellulose degradation capacity.

[0098] (3) Filter paper strip disintegration and antagonism experiment

[0099] Aspirate the bacterial culture (OD) of each purified strain during the logarithmic phase. 540 =0.5) 2 mL was added to 98 mL of filter paper strip culture medium and incubated at 50℃ and 180 r min. -1The filter paper strips were cultured for 7 days under the specified conditions. The degree of degradation of the filter paper was observed by the number of "+" signs. The antagonistic phenomenon between the strains was observed in accordance with the method of Zhang Bizhou et al. (Zhang Bizhou, Hong Bo, Zhang Tao, et al. Study on the compound effect and degradation characteristics of corn straw degrading bacteria under low temperature conditions [J]. Journal of Northern Agriculture, 2021, 49(06): 71-78.).

[0100] (4) Molecular biological identification

[0101] Genomic DNA was extracted from the strain using the Ezup column-based bacterial genomic DNA extraction kit (Shanghai Sangon Biotech Co., Ltd.). Following the method of Yang Chenxian (Yang Chenxian. Screening of lignin-degrading bacteria and study on the mechanism of lignin degradation by their enzymes [D]. Yangling: Northwest A&F University, 2018.), 16S rDNA PCR amplification was performed. After quality control, the product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with ribosome databases, and BLAST homology analysis was performed on the NCBI website. Highly homologous sequences were exported and a phylogenetic tree was constructed using the Neighbor Joining method in MEGA11 software.

[0102] (5) Preparation of microbial agents and determination of enzyme activity on filter paper

[0103] Take the logarithmic period (OD) respectively 540 =0.5) 1 mL of bacterial suspension was added to 100 mL of CMC-Na liquid culture medium and incubated at 50 °C and 180 r / min. -1 The bacterial agent was cultured to the logarithmic phase under certain conditions to prepare the bacterial agent.

[0104] Filter paper enzyme activity (FPA) represents the total enzyme activity of cellulase and can be used to reflect cellulase activity (Wang Jilian, Li Mingyuan, Zhou Qian, et al. Screening of cellulose-degrading bacteria and study on degradation performance of compound bacteria in compost [J]. Journal of Nuclear Agricultural Sciences, 2023, 37(01): 180-187.). The inoculant (OD) 540 =0.5) was added to 100 mL of CMC-Na liquid culture medium at a volume ratio of 2%, and incubated at 50 °C and 180 r min. -1 Cultured in medium for 3 days, with samples taken every 12 hours. Take 5 mL of bacterial culture and incubate at 4℃ and 6000 r / min. -1 Centrifuge for 10 min under the specified conditions, and then take the supernatant to determine the enzyme activity on the filter paper.

[0105] The enzyme activity on filter paper was determined according to the method of Sarula et al. (Sarula, Gao Julin, Yu Xiaofang, et al. Screening of low-temperature degradation complex strains of corn straw [J]. Chinese Agricultural Science, 2013, 46(19): 4082-4090.).

[0106] 1.2.2 Effects of C4 inoculant on the composting of button mushroom residue

[0107] A simulated composting experiment using button mushroom substrate was conducted in an electrically heated constant-temperature incubator for a total of 28 days. The experiment was divided into a control group (CK) and an experimental group (T). The initial C / N ratio was adjusted to 30, the moisture content was 60%, and C4 inoculant was added at 0.5% (vw) of the substrate. -1 The proportions of samples added were as shown in Table 1. The incubator temperatures were set as follows: 53℃ in the first week, 58℃ in the second week, 43℃ in the third week, and 33℃ in the fourth week. All samples were placed in a small reactor (covered but not completely sealed, with dimensions of 30 cm, 20 cm, and 10 cm respectively), and the temperature of the pile was measured at 16:00 daily. An equal amount of water was added periodically to maintain the moisture content of the substrate, and the pile was not turned over throughout the process. 20 g samples were collected at multiple points using the quartering method on days 0, 7, 14, 21, and 28. The obtained samples were dried at 65℃, ground, and sieved through a 0.5 mm sieve before being used to measure various physicochemical properties.

[0108] Table 1 Different treatment groups of mushroom residue compost

[0109]

[0110] Measurement indicators and methods

[0111] Moisture content: Take 5 g of fresh sample, dry it at 65℃ to constant weight, and calculate the moisture content.

[0112] pH and EC: Refer to the experimental method of Liu Linpei et al. (Liu Linpei, Guan Xiuqiong, Wang Hong, et al. Nitrogen changes and composting process of edible fungi residue and liquor lees during co-composting [J]. Food and Fermentation Industries, 2020, 46(21): 188-194.).

[0113] TOC: Measured according to the burning method of Ma Zheng et al. (Ma Zheng, Zhang Bosong, Xu Changying, et al. Analysis of organic matter determination method based on the new standard of organic fertilizer [J]. Jiangxi Journal of Agricultural Sciences, 2013, 25(05): 72-74.).

[0114] Kjeldahl nitrogen (N): determined by the Kjeldahl method (Yue Shilin, Ma Xiaoyong, Jiang Guojun. Effects of high-temperature and low-temperature resistant solid compound microbial agents on cow manure compost [J]. Journal of Animal Ecology, 2024, 45(02): 55-60.).

[0115] Seed germination rate (GI): Seed root length was measured non-destructively according to the NY / T 525-2021 standard and the method of Wang Guoying (Wang Guoying, Yuan Jing, Kong Yilin, et al. Method for determining germination index of compost seeds and screening of sensitive seeds [J]. Transactions of the Chinese Society of Agricultural Engineering, 2021, 37(19): 220-227.).

[0116] Heavy metal content analysis: Weigh 0.200 g of sample into a digestion tube, add 9 mL of 65% nitric acid and 3 mL of hydrofluoric acid, heat at 200℃ for 240 min in a sealed container, remove acid at 150℃ for 120 min with the lid open, and cool to room temperature before using 0.5 mol L⁻¹ -1 The sample was diluted to 50 mL with nitric acid and 100 ppb Au(NO3)3 solution, and the GSD-9 standard material was digested in the same batch to verify the reliability of the experiment. The contents of As, Hg, Pb, Cd, Cr, Cu, Zn and Ni in the sample were determined by inductively coupled plasma mass spectrometry.

[0117] 1.2.3 The impact of compost products on crop seedling growth

[0118] A control group (CK) and experimental groups (T1 and T2) were set up, with substrate ratios shown in Table 2. Each group had five replicates. After 20 days of cultivation, the plant height, stem diameter, total fresh weight, root fresh weight, total dry weight, and crown width of each crop were measured. During the cultivation period, the environmental conditions and cultivation management measures for each treatment were kept consistent. Watering was carried out once a day at 9:00 AM, with 12 hours of light followed by 12 hours of darkness. The light intensity was 20,000 lx, and the temperature was set at 28°C during the day and 20°C at night.

[0119] Table 2 Different treatment groups of crop seedling substrate

[0120]

[0121] Measurement indicators and methods

[0122] Plant height: The distance from the surface of the cultivation substrate to the highest point of the mature crop plant.

[0123] Stem diameter: Measure the stem diameter of the upper, middle and lower parts of the tomato plant, and record the average value as the stem diameter of a single plant.

[0124] Total fresh weight: Weigh the fresh weight of the above-ground parts and roots of the crop after washing (accurate to 0.01g).

[0125] Crown width: The average value of the horizontal and vertical diameters of each crop.

[0126] Dry matter content: Dry matter content = (dry weight of plant / fresh weight of plant) × 100% (Du Yanmei, Xin Guimin, Li Long, et al. Study on screening of pepper seedling substrate based on black fungus residue [J]. Northern Horticulture, 2019, (13): 17-22.).

[0127] 1.3 Data Processing

[0128] Experimental data were plotted and analyzed for differences using SigmaPlot 14.0 and SPSS 26. Differences were considered significant at the p-level (<0.05) and highly significant at the p-level (<0.01). Unless otherwise specified, each experiment was conducted in triplicate.

[0129] 2. Results and Analysis

[0130] 2.1 Preparation of thermophilic cellulose-degrading bacterial agent

[0131] 2.1.1 Primary and secondary screening of cellulose-degrading bacteria

[0132] Congo red staining revealed that all four strains exhibited clear zones of varying sizes, with X3 having the largest and X4 the smallest. The ratio of the clear zone to the strain diameter for all four strains was close to 2.0, indicating strong cellulose degradation capabilities (Table 3).

[0133] Table 3. X3~X6 D / d and its degradation effect on filter paper strips

[0134]

[0135] 2.1.2 Preparation of microbial agents

[0136] After 7 days of cultivation using filter paper strips as the sole carbon source, the degradation effects are shown in Table 3. The X3 bacterial agent showed the best degradation effect on the filter paper strips; the degraded filter paper strips exhibited severe edge damage and obvious degradation traces, and the solution became turbid. The filter paper strips from the X4, X5, and X6 bacterial agents showed no obvious degradation traces, and the solutions were relatively clear. The control group showed intact filter paper strips, and the solution was clear. Based on these two sets of experiments, the X3 strain, with its superior degradation effect, was ultimately determined to be used for the preparation of the C4 bacterial agent.

[0137] The C4 bacterial agent reached its maximum enzyme activity of 16.0 U / mL on filter paper after 24 hours. -1 During the stationary phase of bacterial growth, the enzyme activity on filter paper remained relatively stable. Figure 1 ).

[0138] 2.1.3 Molecular biological identification of strain X3

[0139] After PCR amplification, the DNA sample showed distinct and uniformly bright bands at the 1200bp-1500bp position on agarose gel electrophoresis. The 16S rDNA gene sequence of the strain is shown in SEQ ID NO.1.

[0140] SEQ ID NO.1:

[0141]

[0142] The comparison results from the ribosome database show that strain X3 belongs to the genus Bacillus, specifically Bacillus licheniformis.

[0143] 2.2 Effects of C4 inoculant on the composting of button mushroom residue

[0144] 2.2.1 Changes in temperature and moisture during the composting process

[0145] Changes in temperature and moisture during the composting process, such as Figure 2 As shown. In this application, composting lasted for 28 days. On day 2, the temperature in both the control and experimental groups exceeded 50°C, and this high-temperature period continued for 14 days thereafter. The temperature difference between the two groups was not significant, and the temperature in both treatment groups exceeded 55°C and remained above 3 days, meeting the requirements for eradicating pathogenic microorganisms and ensuring that the sanitary index of compost was met (Meng XY, Dai JL, Zhang Y, et al. Composted biogas residue and spent mushroom substrate as a growth medium for tomato and pepperseedlings [J]. Journal of Environmental Management, 2018, 216: 62-69.). At the end of the experiment, the percentages of the experimental group and the control group were 25.5% and 26.3%, respectively, which met the requirement that the moisture content of organic fertilizers be less than 30% (Jia XJ, Qin XM, Tian XP, et al. Inoculating with the microbial agents to start up the aerobic composting of mushroom residue and wood chips at low temperature[J]. Journal of Environmental Chemical Engineering, 2021, 9(4): 10.).

[0146] 2.2.2 Changes in pH and conductivity during the composting process

[0147] pH value is an important indicator affecting the composting process; both excessively high and low pH levels can adversely affect microbial activity and the rate of organic matter decomposition (Meng XY, Dai JL, Zhang Y, et al. Composted biogas residue and spent mushroom substrate as a growth medium for tomato and pepperseedlings [J]. Journal of Environmental Management, 2018, 216: 62-69.). Figure 3 (A) It can be seen that the pH values ​​in both groups consistently showed an upward trend, which may be because microbial activity converted organic nitrogen into NH4. + The pH value was increased (Zhou LY, Yang XP, Wang XW, et al. Effects of bacterial inoculation on lignocellulose degradation and microbial properties during cowdung composting [J]. Bioengineered, 2023, 14(1): 213-228.). On day 7, the experimental group had a significantly higher pH than the control group, which may be due to the production of more ammonium nitrogen after the addition of microorganisms in the experimental group. As composting continued, the decomposed small molecules continued to volatilize, and the pH tended to stabilize. Finally, the pH values ​​of both compost groups were between 5.5 and 8.5, which met the pH requirements of organic fertilizers (Zhao Y, Zhao Y, Zhang ZC, et al. Effect of thermo-tolerant actinomycetes inoculation on cellulose degradation and the formation of humic substances during composting [J]. Waste Management, 2017, 68: 64-73.).

[0148] The EC value is an indicator of compost substrate salinity, reflecting the total salt concentration of the compost and representing whether the sample has phytotoxicity. From... Figure 3(B) It can be seen that EC gradually decreases with substrate degradation, which is consistent with the results of Li Haijie (Li HJ, Mu RH, He YH, et al. Effect of microbial agents on maturity, humification, and stability and the bacterial succession of spent mushroom substrate composting [J]. Environmental Science and Pollution Research, 2022, 29(58): 87775-87789.). On day 7, the experimental group was significantly lower than the control group, indicating that adding microbial agents is beneficial to reducing the salt content of compost products.

[0149] At the end of composting, the EC values ​​of the CK and T groups were 3.67 mS cm⁻¹. -1 and 3.64 mS cm -1 Their values ​​are all below 4.0 mS cm -1 This indicates that it has no adverse effects on plant growth.

[0150] 2.2.3 Changes in total organic carbon and Kjeldahl nitrogen during the composting process

[0151] TOC is an important carbon and energy source for microorganisms (Jiang JS, Wang Y, Liu J, et al. Exploring the mechanisms of organic matter degradation and methane emission during sewage sludge composting with added vesuvianite: Insights into the prediction of microbial metabolic function and enzymatic activity [J]. Bioresource Technology, 2019, 286: 10.). Figure 4 As shown in (A), the TOC content showed a decreasing trend. Compared with the initial value, the organic matter content of the experimental group decreased by 12% at the end of composting, which was greater than the 9.8% decrease in the control group. Compared with the control, when composting entered the high-temperature stage, the addition of exogenous microbial agents had a greater promoting effect on the reduction of TOC during composting.

[0152] Throughout the composting process, Kjeldahl nitrogen exhibited a pattern of first decreasing and then increasing, such as Figure 4 (B) In the control group, Kjeldahl nitrogen was significantly higher than that in the experimental group on day 7. However, in the later stages of composting, the increase in Kjeldahl nitrogen in the experimental group was more significant on day 21. The early stage of composting is caused by microbial activity leading to the formation and release of ammonia, resulting in a decrease in Kjeldahl nitrogen. The increase in Kjeldahl nitrogen in the later stage of composting may be attributed to the concentration effect of mass loss caused by microbial metabolism (Bao JF, Lv YF, Qv M, et al. Evaluation of key microbial community succession and enzyme activities of nitrogen transformation in pig manure composting process through multi angle analysis [J]. Bioresource Technology, 2022, 362: 10.).

[0153] 2.2.4 Changes in carbon-nitrogen ratio and T value during the composting process

[0154] C / N determination is shown in [reference]. Figure 5 (A) The results showed that the experimental group experienced a greater decrease in C / N ratio during the high-temperature stage than the control group. By the end of the composting process, the C / N ratio of the experimental group was 14.85, which was 14.5% lower than that of the control group, reflecting to some extent the promoting effect of C4 microbial agent on the composting process.

[0155] like Figure 5 As shown in (B), the T values ​​of the experimental group were significantly lower than those of the control group on days 7, 14, 21, and 28. The T value of the experimental group on day 21 was 0.54, which was 22% lower than that of the control group, indicating that the decomposition standard had been met.

[0156] By the end of the composting process, both groups of compost products had matured, with the experimental group maturing about 7 days earlier than the control group.

[0157] 2.2.5 Seed germination index and heavy metal content during composting process

[0158] GI (Growth Index) is an indicator of seed vigor and the most convincing method for assessing compost maturity (Zhang L, Sun XY. Changes in physical, chemical, and microbiological properties during the two-stage co-composting of green waste with spent mushroom compost and biochar [J]. Bioresource Technology, 2014, 171: 274-284.). On day 7 of composting, the GI of the experimental group was significantly lower than that of the control group, see [reference needed]. Figure 6 (A) This may be because after the addition of microbial agents, a large amount of organic matter is decomposed into toxic substances such as high concentrations of ammonia and organic acids. After the high-temperature period, the GI values ​​of both groups increased significantly. By the end of the composting process, the compost products of both groups were fully decomposed, and the GI value of the experimental group was the highest at 90%, which was significantly greater than that of the control group at 83%.

[0159] exist Figure 6 In (B), the average contents of heavy metals As, Hg, Pb, Cd, Cr, Cu, Ni, and Zn in the experimental group were 3.47, 0.03, 11.15, 0.32, 23.78, 38.26, 18.33, and 194.13 mg kg, respectively. -1 The levels of the eight heavy metals in the experimental group and the control group were 23%, 1.5%, 22.3%, 10.7%, 15.9%, 12.75%, 36.7%, and 24.3% of the domestic heavy metal standard limits for organic fertilizers, respectively. There was no significant difference in the content of the eight heavy metals between the experimental group and the control group, indicating high ecological safety.

[0160] 2.3 Impact of compost products on crop seedling growth

[0161] 2.3.1 Changes in seedling height and stem diameter of crops

[0162] Compared to soil, button mushroom substrate compost contains more organic matter and has greater porosity, which, under certain conditions, provides more nutrients and air for crop seedlings. Figure 7 As shown in the left figure, the seedling height of peppers, tomatoes, and chamomiles all showed the order T2 > T1 > CK. Specifically, the pepper seedlings in group T2 were 11.1% taller than those in group CK, the tomato seedlings in group T2 were 17.8% and 15.2% taller than those in groups CK and T1, respectively, and the chamomile seedlings in group T2 were 20.5% taller than those in group CK. This indicates that compost products with added C4 microbial agent are more effective in promoting the growth of crop seedlings than soil cultivation substrates.

[0163] exist Figure 7In the right figure, the diameter of chili seedlings showed the order of T2 > CK > T1. Specifically, the diameter of chili seedlings in group T2 was 8.6% and 12.5% ​​higher than that in groups CK and T1, respectively. For tomato seedlings, the diameter showed the order of T2 > T1 > CK. Specifically, the diameter of tomato seedlings in group T2 was 20.9% and 10.6% higher than that in groups CK and T1, respectively. For chamomile seedlings, the diameter showed the order of T1 > CK > T2, but there was no statistically significant difference in diameter among the three groups.

[0164] Among the three crops, the T2 group of button mushroom residue showed the best growth-promoting effect on tomato seedlings, followed by pepper.

[0165] 2.3.2 Changes in the fresh weight of crop seedlings and aboveground fresh weight

[0166] Depend on Figure 8 As shown in the left figure, in terms of the total fresh weight of pepper, tomato, and chamomile seedlings, the order was T2 > T1 > CK. Among them, the total fresh weight of tomato seedlings in group T2 was 37.2% and 28.7% higher than that in groups CK and T1, respectively. This indicates that the mushroom residue composted by the fungal agent can promote the accumulation of organic matter in tomato plants.

[0167] exist Figure 8 In the right figure, the aboveground fresh weight of pepper seedlings showed T1 > T2 > CK, with no statistically significant difference. The aboveground fresh weight of tomatoes and chamomile seedlings both showed T2 > T1 > CK. Specifically, the aboveground fresh weight of tomato seedlings in group T2 was 34.6% higher than that in group CK, a statistically significant difference.

[0168] The results showed that compost products with added C4 microbial agent could increase the total fresh weight of tomato seedlings and the fresh weight of the aboveground parts, but had no significant promoting effect on peppers and chamomile.

[0169] 2.3.3 Changes in the fresh weight of crop seedling roots and the width of the seedling crown

[0170] Depend on Figure 9 As shown in the left figure, the root fresh weight of both chili peppers and chamomile was T2 > CK > T1, with no statistically significant differences. For tomatoes, the root fresh weight was T2 > T1 > CK. Specifically, the root fresh weight of tomatoes in group T2 increased by 72.4% and 61.8% compared to CK and T1, respectively, with statistically significant differences. This indicates that mushroom substrate compost with added C4 inoculant is more suitable as a cultivation substrate for tomatoes.

[0171] exist Figure 9In the right figure, the canopy width of peppers showed a pattern of T1 > T2 > CK, with the canopy width of peppers in group T2 being 19.7% larger than that of group CK, a statistically significant difference. For tomatoes, the canopy width showed a pattern of T2 > T1 > CK, with the canopy width of tomatoes in group T2 being 29.5% and 13.6% larger than that of groups CK and T1, respectively, both statistically significant differences.

[0172] The results showed that compost products with added C4 bacteria could increase the fresh weight of tomato seedling roots and the crown width, but had no significant effect on peppers and chamomile.

[0173] 2.3.4 Changes in the dry weight and dry matter content of crop seedlings

[0174] Depend on Figure 10 The left figure shows that the dry weight of chili pepper, tomato, and chamomile seedlings was T2 > T1 > CK, and the difference between the tomato T2 group and the T1 and CK groups was significant. Figure 10 In the right figure, the dry matter content of chili peppers showed T1 > T2 > CK, with no statistically significant differences; the dry matter content of tomatoes showed T2 > T1 > CK, with significant differences between the T2 group and the CK and T1 groups; the dry matter content of chamomile showed T1 > CK > T2, with no statistically significant differences.

[0175] The results showed that compost products fermented with C4 microbial agent could promote the increase of dry weight and dry matter content of tomatoes, but had no significant effect on peppers and chamomile.

[0176] 3. Discussion

[0177] This application screened a thermophilic cellulose-degrading bacterium from button mushroom substrate and formulated inoculant C4. Using button mushroom substrate as the main raw material, the effect of adding inoculant C4 on the composting process was investigated. Subsequently, the compost product replaced 50% of the soil for crop seedling cultivation, and the potential of returning the compost product to the field was evaluated. The research results provide a theoretical basis and data support for the fertilizer and substrate utilization of button mushroom substrate.

[0178] Bacteria, fungi, and actinomycetes are currently the main microorganisms that degrade cellulose, with bacteria being the most studied. Cellulose-degrading bacteria mainly include Clostridium, Bacillus, Monotyphi, and Cladosporium (Liang Tao, Hu Liujie, Zhang Tao, et al. Analysis of nutrient and heavy metal content in edible fungi residue in Chongqing [J]. Journal of Plant Nutrition and Fertilizers, 2022, 28(04): 715-725. Li Xin, Li Jianxun, Wang Yumeng, et al. Research progress on microbial degradation of lignocellulose and its feed application [J]. Feed Industry, 2024, 45(09): 137-144). The various types of cellulases produced by the strains of the microbial agent in this application have good complementarity in the degradation of lignocellulose and can synergistically complete the degradation of cellulose in the fungi residue. Wang Xueli et al. (Wang Xueli, Shen Kaiwei, Lei Chao, et al. Screening and degradation characteristics analysis of cellulose-degrading bacteria in waste microbial residue [J]. Journal of Southern Agriculture, 2021, 52(11): 2913-2922.) screened cellulose-degrading bacteria whose filter paper enzyme activity reached a maximum of 33.14 U / mL. -1 However, excessively high enzyme activity accelerates the degradation of lignocellulose but hinders the preservation of organic matter in the compost. For mushroom residue composting, inoculants with specific enzyme activities should be selected to achieve both nutrient preservation and accelerated decomposition. The thermophilic cellulose-degrading inoculant developed in this application can provide high-quality inoculum resources for the decomposition of button mushroom residue.

[0179] Based on the experimental composting scale of this application, urea, which has a relatively high nitrogen content compared to animal manure such as chicken manure, releases nitrogen quickly, and is easily decomposed and utilized by microorganisms, was chosen as the nitrogen source. In this application, high temperature was maintained for 14 days. Compared with the control group, C4 bacteria accelerated the consumption of organic matter, rapidly reduced the T value, accelerated the maturity of compost, reduced product salinity, and increased the GI value and nitrogen content. The heavy metal content of both groups was at a low level, meeting the standards for organic fertilizers and demonstrating strong safety. In practice, the inoculant can be mixed with mushroom substrate at 0.5% by mass and piled into a windrow-shaped pile with a height of 1-2 m for fermentation. A plastic film is added on top of the pile to maintain temperature. The fermentation stage of the compost is determined by daily monitoring of the pile temperature. Once certain physicochemical indicators are met, the compost is considered to be fully matured.

[0180] The results of this application indicate that button mushroom mycelium compost does not promote the growth of all crop seedlings, and different compost products have different effects on crop seedlings. This may be related to the quality and composition of the compost products, as well as the different root systems' adaptability to the environment. In this study, the C4 inoculant may have accelerated the degradation of cellulose in the cultivation substrate, and the resulting small molecules facilitated better colonization of rhizosphere microorganisms. The results show that button mushroom mycelium composted with C4 inoculant improved the fresh quality of tomato seedling roots and had a significant promoting effect on tomato seedling growth, but its effect on pepper and chamomile seedlings was not significant. Using 50% button mushroom mycelium compost helps improve the growth status of tomato seedlings.

[0181] 4. Conclusion

[0182] This application uses button mushroom residue as the research object, screens thermophilic cellulose-degrading bacteria to prepare inoculant, and uses urea as the nitrogen source to explore the effect of the inoculant on shortening composting time and improving the quality of compost products. A pot experiment was conducted to replace 50% of the soil with the decomposed compost product in crop seedlings to evaluate the feasibility of large-scale application of button mushroom residue compost products to the field. The main results and conclusions of the study are as follows:

[0183] (1) The selected strain is Bacillus licheniformis. It has the characteristics of high temperature resistance, strong adaptability and spore production. The bacterial agent (C4) prepared by the strain showed stable filter paper enzyme activity.

[0184] (2) By the end of the composting process, the total organic carbon and electrical conductivity (EC) values ​​of the compost products in the experimental group decreased by 14.7% and 0.8% respectively compared with the control group, and the GI value was also significantly higher than that of the control group. The addition of C4 microbial agent can significantly improve the biological activity during the composting process and reduce plant toxicity. The composting time was shortened by about one week. The heavy metal content of the compost products in both groups was far below the industry standard threshold, which has little environmental harm and high compost safety.

[0185] (3) In the cultivation experiment, the plant height, fresh weight, and dry weight of the three plants were all in the order T2 > T1 > CK. The addition of C4 microbial agent could promote the biomass accumulation of seedlings; however, in terms of stem diameter, crown width, and root weight, except for tomato plants, the experimental group did not achieve a significant advantage over the control group. The compost products of C4 microbial agent had a relatively stable promoting effect on the growth of tomato plants, but the promoting effect on peppers and chamomile was not obvious.

[0186] In summary, C4 microbial agent can shorten the composting cycle, improve composting efficiency and product quality, and meet the requirements for harmlessness. Compost products matured with C4 microbial agent have higher safety and product quality, and can replace 50% of soil for crop seedling cultivation. The results of this study have important reference value for improving the comprehensive utilization level of button mushroom residue resources.

[0187] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0188] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. Bacillus licheniformis X3, characterized in that, The preservation number is GDMCC No: 66520.

2. A fermentation inoculant characterized in that, The fermentation agent comprises the Bacillus licheniformis X3 of claim 1.

3. A method for preparing a fermentation inoculum, characterized by, The preparation method comprises the following steps: inoculating the Bacillus licheniformis X3 of claim 1 into a fermentation medium, culturing, and preparing a bacterial agent.

4. The method of producing the fermentation inoculant according to claim 3, characterized by, The culturing satisfies one or more of the following conditions: (1) the fermentation medium comprises a CMC-Na liquid medium; (2) the temperature is 45-55°C; (3) rotation speed of 170 r min -1 190 r min -1 ; (4) the culturing is performed until the Bacillus licheniformis X3 is in the logarithmic phase. The Bacillus licheniformis X3 in the logarithmic phase is inoculated.

5. The method of producing the fermentation inoculant according to any one of claims 3 to 4, characterized in that, The preparation method comprises the following steps:

6. A method for producing an organic fertilizer, characterized by, The fermentation agent prepared by the Bacillus licheniformis X3 of claim 1, the fermentation agent of claim 2, or the preparation method of any one of claims 3-5 is used to ferment Agaricus bisporus residue to prepare an organic fertilizer. The organic fertilizer is prepared by composting.

7. The method of claim 6, wherein the organic fertilizer is prepared by mixing the organic material and the inorganic material in a ratio of 1: 1 to 1:

3. The composting satisfies one or more of the following conditions:

8. The method of claim 7, wherein the organic fertilizer is prepared by mixing the organic material and the inorganic material in a ratio of 1: 1 to 1:

3. (1) the ratio of the fermentation agent to the Agaricus bisporus residue is (8-12) mL:(1800-2200) g; (2) the C / N ratio of the initial system of the composting is 20-30; (3) the composting time is 7-28 days; and (4) the composting process does not involve turning; Optionally, urea is used as a nitrogen source to adjust the C / N of the initial system of the composting. Optionally, the ratio of the fermentation agent, the urea, and the Agaricus bisporus residue is (8-12) mL:(20-28) g:(1800-2200) g. The preparation method of any one of claims 6-8 is used.

9. An organic fertilizer, characterized by, The planting method uses the organic fertilizer of claim 9 in the planting of crops; 10. A method for growing a crop, characterized by, Optionally, the crops comprise vegetables; Optionally, the vegetables comprise one or more of tomatoes and peppers. ​

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