Microbial agent and application thereof in field of fast decomposition of tail vegetables

Abstract

The application relates to the technical field of tail-cabbage waste resource utilization, in particular to a microbial agent and application thereof in the field of tail-cabbage fast rot. The microbial agent comprises Bacillus pumilus and Bacillus subtilis; the Bacillus subtilis is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 33122; the Bacillus pumilus is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 33121. The application also provides application of the microbial agent in the field of tail-cabbage fast rot. The application widens the tail-cabbage fast rot treatment method, enriches the microbial species that can be used for tail-cabbage fast rot, and can effectively improve the tail-cabbage rotting rate and the yield of the next crop by using the microbial agent provided by the application to carry out tail-cabbage fast rot.

Description

Technical Field

[0001] This application relates to the technical field of resource utilization of vegetable waste, and in particular to a microbial agent and its application in the field of rapid composting of vegetable waste. Background Technology

[0002] Vegetable waste, also known as vegetable scraps, refers to the waste generated at every stage of vegetable production, from harvesting and transportation to shelf placement. This includes unusable parts removed during the harvesting of mature, fresh vegetables, as well as roots left in the soil (field-grown waste); damaged parts that need to be removed during transportation from the field to the market; and parts removed during simple processing to improve the appearance of the vegetables before they are displayed. Studies have shown that vegetable waste accounts for more than 30% of total vegetable production. With the booming development of the vegetable farming industry, the amount of vegetable waste generated is increasing daily.

[0003] Currently, the main methods for handling vegetable waste include direct disposal, landfill, simple composting, chemical treatment, and biological treatment. Direct disposal not only occupies a large amount of land resources but also causes the decaying waste to breed harmful microorganisms, emit foul odors, and pollute the surrounding environment, negatively impacting soil, water, and air. While landfilling can alleviate environmental pressure to some extent, vegetable waste decomposes slowly and easily produces leachate, polluting soil and groundwater. Simple composting suffers from a long composting period and low efficiency, making it unsuitable for large-scale vegetable waste treatment. Chemical treatment can accelerate composting but may introduce chemical residues, affecting soil quality and subsequent crop growth.

[0004] Biological treatment primarily utilizes microbial agents, which can rapidly decompose organic matter in vegetable waste through the metabolic activities of specific microorganisms, significantly shortening the composting cycle and improving processing efficiency, thus meeting the urgent needs of large-scale vegetable waste treatment. However, the application of traditional microbial agents in the field of vegetable waste composting also has shortcomings such as poor adaptability and limited decomposition capacity.

[0005] Based on the above, in order to further improve the application of microbial agents in the field of vegetable waste composting, finding more effective microorganisms for vegetable waste composting has become an urgent problem to be solved. Summary of the Invention

[0006] This application provides a microbial inoculant and its application in the field of rapid composting of vegetable waste. This application broadens the methods for rapid composting of vegetable waste, enriches the types of microorganisms that can be used for this purpose, and utilizes the microbial inoculant provided in this application to effectively increase the composting rate of vegetable waste, while also effectively increasing the yield of the next crop.

[0007] In a first aspect, the present application provides a Bacillus subtilis, which adopts the following technical scheme:

[0008] The Bacillus subtilis provided in the present application was preserved in the China General Microbiological Culture Collection Center on December 18, 2024, and the preservation number is CGMCC No. 33122.

[0009] The above-mentioned Bacillus subtilis is isolated from a rotten baby bok choy sample.

[0010] A plurality of reference strain sequences are obtained from the NCBI (GenBank) database, and the isolated strain and the reference strain are analyzed by using software BioEdit and MEGA11 to construct a phylogenetic tree of the isolated strain and the reference strain, so as to determine that the strain system of the above-mentioned isolated strain is Bacillus subtilis.

[0011] The above-mentioned Bacillus subtilis is used in combination with Bacillus pumilus with preservation number CGMCC No. 33121 for tailing vegetable waste decomposition, which can effectively improve the tailing vegetable waste decomposition rate.

[0012] In a second aspect, the present application provides a culture. The culture includes the above-mentioned Bacillus subtilis.

[0013] In the present application, the culture includes fermentation broth and fermentation supernatant.

[0014] In a third aspect, the present application provides fermentation broth. The fermentation broth includes the above-mentioned Bacillus subtilis.

[0015] In a fourth aspect, the present application provides a bacterial suspension. The bacterial suspension includes the above-mentioned Bacillus subtilis.

[0016] In a fifth aspect, the present application provides a microbial inoculant, which adopts the following technical scheme:

[0017] A microbial inoculant, which includes Bacillus pumilus and Bacillus subtilis.

[0018] The Bacillus subtilis is preserved in the China General Microbiological Culture Collection Center on December 18, 2024, and the preservation number is CGMCC No. 33122.

[0019] The Bacillus pumilus is preserved in the China General Microbiological Culture Collection Center on December 18, 2024, and the preservation number is CGMCC No. 33121.

[0020] The above-mentioned Bacillus pumilus is isolated from a rotten baby bok choy sample. The colony is grayish white with smooth and neat edges. The cell body is rod-shaped and relatively short. The individual morphology of the cell is relatively simple without filamentous structure.

[0021] A plurality of reference strain sequences are obtained from the NCBI (GenBank) database. The isolated strain and the reference strain are analyzed by using software BioEdit and MEGA11 to construct a phylogenetic tree of the isolated strain and the reference strain, so as to determine that the strain of the above-mentioned isolated strain is Bacillus pumilus.

[0022] The above-mentioned Bacillus pumilus is used for tailing vegetable disintegration, which can effectively improve the tailing vegetable disintegration rate. Meanwhile, the above-mentioned Bacillus pumilus and the above-mentioned Bacillus subtilis are used in combination for tailing vegetable disintegration, which can further effectively improve the tailing vegetable disintegration rate.

[0023] Optionally, the effective viable count of the Bacillus subtilis in the microbial inoculant is (6-7)×10 9 CFU / g.

[0024] Optionally, the effective viable count of the Bacillus subtilis in the microbial inoculant is 6.5×10 9 CFU / g.

[0025] Optionally, the effective viable count of the Bacillus pumilus in the microbial inoculant is (5-6)×10 9 CFU / g.

[0026] Optionally, the effective viable count of the Bacillus pumilus in the microbial inoculant is 5.4×10 9 CFU / g.

[0027] In a sixth aspect, the present application provides an application of the above-mentioned Bacillus subtilis, culture, fermentation broth, microbial inoculant and tailing vegetable fast disintegration.

[0028] Optionally, the Bacillus subtilis and the Bacillus pumilus are used in combination, and the tailing vegetable disintegration rate can be improved by more than 13%.

[0029] In summary, the present application includes at least one of the following beneficial technical effects:

[0030] 1. The present application provides a microbial inoculant comprising Bacillus pumilus and Bacillus subtilis.

[0031] The Bacillus subtilis (Bacillus subtilis) was preserved in the China General Microbiological Culture Collection Center on December 18, 2024, and the preservation number is CGMCC No. 33122.

[0032] The Bacillus pumilus is preserved in the China General Microbiological Culture Collection Center on December 18, 2024, and the preservation number is CGMCC No. 33121.

[0033] 2. The microbial agent composed of Bacillus pumilus and Bacillus subtilis is used for decomposition of the overripe vegetables, which can further effectively improve the decomposition rate of the overripe vegetables, increase the content of soil organic matter, nutrient elements and beneficial microorganisms, improve the soil aggregate structure, and be beneficial to improving the yield of the next crop.

[0034] 3. The application expands the treatment method of overripe vegetable fast decomposition, enriches the microbial species that can be used for overripe vegetable decomposition, and can effectively improve the decomposition rate of overripe vegetables and effectively improve the yield of the next crop by using the microbial agent provided in the application for overripe vegetable decomposition. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is the growth state of Bacillus pumilus in the application cultured on beef extract peptone medium for 48 h.

[0036] Figure 2 It is the growth state of Bacillus pumilus in the application cultured on beef extract peptone medium for 48 h.

[0037] Figure 3 It is a result comparison chart of the blank group and the test group in the degradation effect verification of peppers (A chart is the control group-pure water; B chart is the test group-microbial agent).

[0038] Figure 4 It is a result comparison chart of the blank group and the test group in the degradation effect verification of tomatoes (A chart is the control group-pure water; B chart is the test group-microbial agent).

[0039] Figure 5 It is a result comparison chart of the blank group and the test group in the degradation effect verification of baby corn when cultured for 10 d (A chart is the control group-pure water; B chart is the test group-microbial agent).

[0040] Figure 6 It is a result comparison chart of the blank group and the test group in the degradation effect verification of baby corn when cultured for 15 d (A chart is the control group-pure water; B chart is the test group-microbial agent).

[0041] Figure 7 It is the detection result of the yield of lettuce under the next crop in different groups in the open field vegetable overripe vegetable microbial decomposition and field test. DETAILED DESCRIPTION

[0042] Before describing embodiments of the application in detail, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only. Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0043] It should be noted that the terms "first", "second" are used only for the purpose of description, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined as "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise stated.

[0044] The endpoints of the ranges and any values disclosed in the present application are not limited to the precise values stated. The ranges or values should be construed to be roughly about the ranges or values. For ranges of values, the endpoints of the ranges are included in the ranges, the endpoints of the ranges and the individual points are included in the ranges, and the individual points are included in the ranges, which should be considered to be specifically disclosed herein.

[0045] In the present application, the term "comprising" or "including" is an open-ended expression, i.e. including the contents indicated in the present application, but not excluding other contents.

[0046] The present application provides a microbial inoculant, which comprises Bacillus subtilis with a preservation number of CGMCC No.33122 and Bacillus pumilus with a preservation number of CGMCC No.33121. The microbial inoculant provided by the present application is used in the field of fast decomposition of tail vegetables, which can effectively improve the decomposition rate of tail vegetables. The tail vegetables can be all waste branches and rotten leaves of vegetables known in the art. For example, the vegetables include peppers, tomatoes, baby corns, broccoli, etc.

[0047] In order to make the purpose, technical solutions and advantages of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application. The embodiments described below are exemplary and are used to explain the present application, but cannot be understood as limiting the present application.

[0048] Unless otherwise specified, the techniques or conditions in the embodiments are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the reagents or instruments used are all conventional products that can be obtained from the market.

[0049] The reagents, solvents and other test materials used in the following examples can be commercially available.

[0050] In the following examples, the formula of LB solid medium is as follows: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar in 1 L of volume; the formula of LB liquid medium is as follows: 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 1 L of volume.

[0051] The application will be further described in detail in combination with examples, drawings and test results.

[0052] Example

[0053] Example 1 - Screening of strains

[0054] The present example provides a screening process of a new bacterial species. Specifically, the process comprises the following steps:

[0055] (1) Sample collection: the sample is selected from rotten baby bok choy (rotten baby bok choy obtained by placing the baby bok choy left over after picking from a field where baby bok choy is planted for a long time at room temperature for 7 days).

[0056] (2) Preliminary isolation of target bacterial species: 5 g of the collected sample is weighed and added to a pre-sterilized triangular flask containing 500 ml of physiological saline under sterile conditions, and then fully shaken at room temperature for 30 min. After that, the sample is diluted to 10 -5 , 10 -3 , 10 -4 , and 10 -5 , respectively, 0.1-0.2 ml of each dilution is taken and spread on cellulose Congo red medium plates (formula: 1 g / L of sodium nitrate, 1.2 g / L of disodium hydrogen phosphate, 0.9 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium sulfate, 0.5 g / L of potassium chloride, 0.5 g / L of yeast extract powder, 0.5 g / L of acid hydrolysis casein, 0.2 g / L of Congo red, 5 g / L of cellulose powder, and 15 g / L of agar; pH value is 7.0±0.1 at 25°C), and then the spread cellulose Congo red medium plates are placed at 28°C for inverted culture for 48 h.

[0057] After culture, two strains produce transparent circles. The colonies producing transparent circles are picked and streaked on LB solid medium plates for isolation and purification (cultured according to the above culture conditions) for 2-3 times until the colonies are single, indicating that the isolation and purification are completed; and then the colonies are stored for use.

[0058] (3) Preliminary identification: the strains obtained by isolation and purification (i.e., isolated strains) are observed under a microscope. According to the observation results, it is preliminarily determined that both strains are Bacillus.

[0059] Example 2

[0060] This embodiment provides the single-strain properties and sequencing results of an isolated strain obtained in Example 1. Details are as follows:

[0061] (I) Properties of Single Strains

[0062] (1) Morphological characteristics

[0063] Isolated strain: Colonies are grayish-white with smooth, regular edges, as shown in the results. Figure 1 As shown. Figure 1 To determine the growth status of the isolated strain after 48 hours of culture on beef extract peptone medium, the specific formulation of the beef extract peptone medium is as follows: per 1L of distilled water, there are 3g of beef extract, 10g of peptone, 5g of sodium chloride, and 15-20g of agar, with a pH of 7.0 and a culture temperature of 30℃.

[0064] (2) Cultivation characteristics

[0065] The optimal growth conditions for this strain are: pH 6.6-7.0 and temperature 4-30℃.

[0066] (3) Functional characteristics

[0067] Using the above-mentioned isolated strains for the decomposition of vegetable waste can effectively improve the decomposition rate of vegetable waste.

[0068] (II) Sequencing

[0069] The isolated strains were sent to a third-party company for sequencing to obtain their 16S rRNA. Multiple reference strain sequences were obtained from the NCBI (GenBank) database. The 16S rRNA of the isolated strains and the reference strains were analyzed using BioEdit and MEGA11 software, and a phylogenetic tree of the isolated strains and reference strains was constructed. This confirmed that the strain lineage of the isolated strains was *Bacillus pumilus*.

[0070] Compared with related technologies, the strain isolated in this application has the following effects: Experimental results show that when the isolated strain is used to decompose chili peppers, tomatoes, and baby bok choy, the decomposition rates of the waste products can reach 85.83%, 87.66%, and 85.83%, respectively. Therefore, the *Bacillus pumilus* obtained in this application can effectively improve the decomposition rate of waste products.

[0071] Bacillus pumilus was deposited on December 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33121. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0072] Example 3

[0073] The present example provides a fermentation broth of Bacillus pumilus.

[0074] The preparation method of the fermentation broth is as follows:

[0075] The isolated strain obtained in Example 1 was activated and inoculated into LB liquid medium with pH 7, and cultured in a shaker at 34°C and 150 r / min for 24 h to obtain a fermentation broth of Bacillus pumilus.

[0076] Example 4

[0077] The present example provides a fermentation supernatant of Bacillus pumilus.

[0078] The preparation method of the fermentation supernatant is as follows:

[0079] The isolated strain obtained in Example 1 was activated and inoculated into LB liquid medium with pH 7, and cultured in a shaker at 34°C and 150 r / min for 24 h to obtain a fermentation broth of Bacillus pumilus; the fermentation broth was centrifuged, and the supernatant was retained to obtain a fermentation supernatant of Bacillus pumilus.

[0080] Example 5

[0081] The present example provides a bacterial suspension of Bacillus pumilus.

[0082] The preparation method of the bacterial suspension is as follows:

[0083] The isolated strain obtained in Example 1 was activated and inoculated into LB liquid medium with pH 7, and cultured in a shaker at 34°C and 150 r / min for 24 h to obtain a fermentation broth of Bacillus pumilus; the fermentation broth was centrifuged, and the precipitate was resuspended with sterile normal saline to obtain a bacterial suspension of Bacillus pumilus.

[0084] Example 6

[0085] The present example provides the single strain properties and sequencing results of another isolated strain obtained in Example 1. The details are as follows:

[0086] (1) Single strain properties

[0087] (1) Morphological characteristics

[0088] Isolated strain: The color of the colony was white, round, opaque, flat and dry. The results are shown in Table 1. Figure 2 Figure 2 ​To observe the growth status of the isolated strain after 48 hours of culture on beef extract peptone medium, the specific formulation of the beef extract peptone medium is as follows: per 1L of distilled water, there are 3g of beef extract, 10g of peptone, 5g of sodium chloride, and 15-20g of agar, with a pH of 7.4-7.6 and a culture temperature of 30℃.

[0089] (2) Cultivation characteristics

[0090] The optimal growth conditions for this strain are: pH = 7.0-7.2, temperature 34-36℃.

[0091] (3) Functional characteristics

[0092] When the aforementioned isolated strain is used in combination with *Bacillus pumilus* with accession number CGMCC No. 33121, the decomposition rate of *Bacillus pumilus* waste products can be effectively improved. Therefore, although the aforementioned isolated strain alone has almost no effect on the decomposition rate of waste products, it can promote and increase the decomposition rate of *Bacillus pumilus* waste products. Therefore, this application provides a microbial inoculant comprising the aforementioned isolated strain and *Bacillus pumilus* with accession number CGMCC No. 33121. Compared with the prior art, this microbial inoculant can effectively improve the decomposition rate of waste products.

[0093] (II) Sequencing

[0094] The isolated strains were sent to a third-party company for sequencing to obtain their 16S rRNA. Multiple reference strain sequences were obtained from the NCBI (GenBank) database. The 16S rRNA of the isolated strains and the reference strains were analyzed using BioEdit and MEGA11 software, and a phylogenetic tree of the isolated strains and reference strains was constructed. This confirmed that the strain lineage of the isolated strains was *Bacillus subtilis*.

[0095] Compared with using Bacillus subtilis (CGMCC No. 33121) alone, the strain isolated in this application has the following effects: Experimental results show that when the microbial inoculant composed of the above-isolated strain and Bacillus subtilis (CGMCC No. 33121) is used for the decomposition of chili peppers, tomatoes, and baby bok choy, the decomposition rates of the vegetable waste can reach 89.74%, 89.10%, and 91.72%, respectively. Therefore, the microbial inoculant containing Bacillus subtilis and Bacillus subtilis provided in this application can effectively improve the decomposition rate of vegetable waste.

[0096] The Bacillus subtilis is preserved in the China General Microbiological Culture Collection Center on December 18, 2024, with a preservation number of CGMCC No. 33122, and a preservation address of No. 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology.

[0097] Example 7

[0098] The present example provides a fermentation liquid of Bacillus subtilis.

[0099] The preparation method of the fermentation liquid is as follows:

[0100] The isolated strain obtained in Example 1 is activated and transferred to an LB liquid medium with a pH of 7.2, and is cultured in a shaking incubator at 36°C and 150 r / min for 24 h to obtain a fermentation liquid of Bacillus subtilis.

[0101] Example 8

[0102] The present example provides a fermentation supernatant of Bacillus subtilis.

[0103] The preparation method of the fermentation supernatant is as follows:

[0104] The isolated strain obtained in Example 1 is activated and transferred to an LB liquid medium with a pH of 7.2, and is cultured in a shaking incubator at 36°C and 150 r / min for 24 h to obtain a fermentation liquid of Bacillus subtilis. The fermentation liquid is centrifuged, and the supernatant is left to obtain a fermentation supernatant of Bacillus subtilis.

[0105] Example 9

[0106] The present example provides a bacterial suspension of Bacillus subtilis.

[0107] The preparation method of the bacterial suspension is as follows:

[0108] The isolated strain obtained in Example 1 is activated and transferred to an LB liquid medium with a pH of 7.2, and is cultured in a shaking incubator at 36°C and 150 r / min for 24 h to obtain a fermentation liquid of Bacillus subtilis. The fermentation liquid is centrifuged, and the precipitate is left and resuspended in sterile physiological saline to obtain a bacterial suspension of Bacillus subtilis.

[0109] Example 10

[0110] The present example provides a microbial inoculant.

[0111] The preparation method of the microbial inoculant is as follows:

[0112] The microbial inoculant was prepared by mixing the bacterial suspension (OD600=1) of Bacillus pumilus with the accession number CGMCC No. 33121 obtained in Example 5 and the bacterial suspension (OD600=1) of Bacillus subtilis with the accession number CGMCC No. 33122 obtained in Example 9 in a volume ratio of 1:1. In the microbial inoculant, the effective bacteria content of Bacillus pumilus was 5.4×10 9 CFU / g, and the effective bacteria content of Bacillus subtilis was 6.5×10 9 CFU / g.

[0113] Performance detection test

[0114] (1) The degradation effect verification on peppers

[0115] (1) The preparation process of the test sample is as follows: the peppers cut into 1-2 cm are evenly divided into 6 groups, 50 g in each group and placed in plastic cups, inoculated with different test objects according to the inoculation amount of 10% (V / V), and placed in a 35°C incubator for incubation. After 15 days, the pepper rot rate was determined.

[0116] The test objects of each group are as follows:

[0117] Inoculation of the microbial inoculant provided in Example 10 (the effective bacteria content of Bacillus pumilus was 5.4×10 9 CFU / g, and the effective bacteria content of Bacillus subtilis was 6.5×10 9 CFU / g) as the test group;

[0118] Inoculation of Bacillus pumilus provided in Example 5 (the effective bacteria content was 5.4×10 9 CFU / g) as the control group one;

[0119] Inoculation of Bacillus subtilis provided in Example 9 (the effective bacteria content was 6.5×10 9 CFU / g) as the control group two;

[0120] Inoculation of Streptomyces albidoflavus (CGMCC No. 12136 in the related art, the effective bacteria content was 5.8×10 10 CFU / g) as the control group three;

[0121] Inoculation of Bacillus tequilensis (CGMCC No. 22956 in the related art, the effective bacteria content was 6.2×10 10 CFU / g) as the control group four;

[0122] Inoculation of pure water as the blank group.

[0123] (2) The calculation method of the tailing silage rate is as follows:

[0124] Tailing silage rate = (initial tailing dry mass (g) - tailing dry mass (g) at the end of cultivation) / initial tailing dry mass (g) x 100%.

[0125] (3) Test results

[0126] The test results are shown in Table 1. The comparison of the results of the test group and the blank group is shown in Table 1. Figure 3

[0127] Table 1: Degradation effect on peppers

[0128]

[0129] As shown in Table 1, the Bacillus pumilus obtained by the application is used for pepper decomposition, and the tailing silage rate can reach 85.83%. It shows that the Bacillus pumilus provided by the application can effectively improve the tailing silage rate.

[0130] As shown in Table 1 and Figure 3 , the microbial agent obtained by the application is used for pepper decomposition, and the tailing silage rate can reach 89.74%, which is significantly improved by 13.71% compared with the related art.

[0131] (II) Degradation effect verification on tomatoes

[0132] (1) The preparation process of the test sample is as follows: the chopped tomatoes of 1-2 cm are evenly divided into 6 groups, each group of 50 g is placed in a plastic cup, inoculated with different test objects according to the inoculation amount of 10% (V / V), and placed in a 35℃ incubator for static culture. After 30 days, the tomato silage rate is determined.

[0133] The test objects of each group are as follows:

[0134] Inoculating the microbial agent (the effective bacteria content of Bacillus pumilus is 5.4 x 10 9 CFU / g, and the effective bacteria content of Bacillus subtilis is 6.5 x 10 9 CFU / g) provided in Example 10 is the test group;

[0135] Inoculating the Bacillus pumilus (the effective bacteria content is 5.4 x 10 9 CFU / g) provided in Example 5 is the control group one;

[0136] Inoculating the Bacillus subtilis (the effective bacteria content is 6.5 x 10 9 CFU / g) provided in Example 9 is the control group two;

[0137] ​Streptomyces albidoflavus (CGMCC No. 12136) of the related art, with an effective bacteria content of 5.8 x 10 10 CFU / g) was the control group three;

[0138] Bacillus tequilensis (CGMCC No. 22956) of the related art, with an effective bacteria content of 6.2 x 10 10 CFU / g) was the control group four;

[0139] The blank group was inoculated with pure water.

[0140] (2) The calculation method of the tail meal decomposition rate is as follows:

[0141] Tail meal decomposition rate = (initial tail meal dry mass (g) - tail meal dry mass (g) at the end of the culture) / initial tail meal dry mass (g) x 100%.

[0142] (3) Test results

[0143] The test results are shown in Table 2. The comparison of the results of the test group and the blank group is shown in Table 2. Figure 4

[0144] Table 2: degradation effect on tomatoes

[0145]

[0146] As can be seen from Table 2, the Bacillus pumilus obtained by the application is used for tomato decomposition, and the tail meal decomposition rate can reach 87.66%. It is shown that the Bacillus pumilus provided by the application can effectively improve the tail meal decomposition rate.

[0147] As can be seen from Table 2 and Figure 4 , the microbial agent obtained by the application is used for tomato decomposition, and the tail meal decomposition rate can reach 89.10%, which is significantly improved by 16.59% compared with the related art.

[0148] (Three) degradation effect verification on baby bok choy

[0149] (1) The preparation process of the test sample is as follows: the chopped baby bok choy of 1-2 cm is evenly divided into 6 groups, each group of 50 g is placed in a plastic cup, different test objects are inoculated according to the inoculation amount of 10% (V / V), and is placed in a 35℃ incubator for static culture. After 10d, 15d, the baby bok choy decomposition rate is determined.

[0150] The test objects of each group are as follows:

[0151] ​The microbial inoculant (Bacillus pumilus with an effective bacteria content of 5.4 x 10 9 CFU / g, Bacillus subtilis with an effective bacteria content of 6.5 x 10 9 CFU / g) provided in Example 10 was used as the test group.

[0152] The Bacillus pumilus provided in Example 5 (with an effective bacteria content of 5.4 x 10 9 CFU / g) was used as the control group 1.

[0153] The Bacillus subtilis provided in Example 9 (with an effective bacteria content of 6.5 x 10 9 CFU / g) was used as the control group 2.

[0154] The Streptomyces albidoflavus (CGMCC No. 12136) provided in the related art (with an effective bacteria content of 5.8 x 10 10 CFU / g) was used as the control group 3.

[0155] The Bacillus tequilensis (CGMCC No. 22956) provided in the related art (with an effective bacteria content of 6.2 x 10 10 CFU / g) was used as the control group 4.

[0156] The blank group was inoculated with pure water.

[0157] (2) The calculation method of the tailing silage decomposition rate is as follows:

[0158] Tailing silage decomposition rate = (initial tailing silage dry mass (g) - tailing silage dry mass at the end of cultivation (g)) / initial tailing silage dry mass (g) x 100%.

[0159] (3) Detection results

[0160] The detection results are shown in Tables 3 and 4. The comparison of the results of the test group and the blank group is shown in Figure 5 and Figure 6 .

[0161] Table 3: Degradation effect on baby bok choy (1)

[0162]

[0163]

[0164] Table 4: Degradation effect on baby bok choy (2)

[0165]

[0166] As can be seen from Tables 3 and 4, the Bacillus pumilus obtained in the present application is used for decomposing Brassica oleracea, and the decomposition rate of tail vegetables after 10 days of culture can reach 86.90%, and the decomposition rate of tail vegetables after 15 days of culture can reach 85.83%. It shows that the Bacillus pumilus provided in the present application can effectively improve the decomposition rate of tail vegetables.

[0167] In combination with Tables 3-4 and Figures 5-6 It can be seen that the microbial agent obtained in the present application is used for decomposing tomatoes, and the decomposition rate of tail vegetables after 10 days of culture can reach 88.05%, and the decomposition rate of tail vegetables after 15 days of culture can reach 91.72%. Compared with the related art, the decomposition rate after 10 days of culture is increased by 23.96%, and the decomposition rate after 15 days of culture is increased by 16.21%.

[0168] (IV) Microbial decomposition of open field vegetable tail vegetables

[0169] (1) Test design

[0170] The test was carried out on July 22, 2023 in Yongchang Experimental Station (East longitude 101°04', North latitude 37°47') for open field vegetable tail vegetable microbial decomposition and field test.

[0171] The test material is broccoli tail vegetables, and its physicochemical properties are shown in Table 5.

[0172] Table 5 Physicochemical properties of broccoli tail vegetables

[0173] Group N (mg / kg) P (mg / kg) K (mg / kg) Moisture content (%) Brussels sprouts tops (dry) 3.44 0.33 3.61 - Brussels sprouts tops (fresh) 1.25 0.12 1.31 63.75％

[0174] Fresh tail vegetables were used for the test. The test was designed for 3 groups, 3 parallel tests for each group, a total of 9 treatments, and the area of each treatment was 18m 2 . Each group is as follows:

[0175] A. Blank group (NC): only routine fertilization, without broccoli tail vegetable field operation. Specifically: take 0.5t / acre organic fertilizer (commercial organic fertilizer 500kg / acre (organic matter 30%, total nutrient 4%), ternary compound fertilizer (21-6-13) 30kg / acre) evenly spread on the soil surface, and use a rotary cultivator to spin the organic fertilizer into the soil, rotary tillage to about 20cm; on July 24, plant the next crop lettuce.

[0176] B. Control group (CK): routine fertilization was carried out at the same time, and brassica napus tail meal was returned to the field; specifically, the brassica napus tail meal was crushed to 3-5 cm, and the crushed brassica napus tail meal was evenly spread on the soil surface, 0.5 t / mu of organic fertilizer (500 kg / mu of commercial organic fertilizer (organic matter 30%, total nutrient 4%), 30 kg / mu of ternary compound fertilizer (21-6-13)) was evenly applied on the surface of the crushed brassica napus tail meal (2240 kg / mu), and the brassica napus tail meal and organic fertilizer were spun into the field with a rotary cultivator, and the brassica napus tail meal was rotary cultivated to about 20 cm; the lower crop lettuce was planted on July 24.

[0177] C. Test group (RM): routine fertilization (reduction of 10%) was carried out at the same time, and brassica napus tail meal was returned to the field, and the microbial inoculant provided in Example 10 (2 billion / mL) was applied in an amount of 0.5 kg / mu. Specifically, the brassica napus tail meal was crushed to 3-5 cm, and the crushed brassica napus tail meal (2240 kg / mu) was evenly spread on the soil surface, 0.5 t / mu of organic fertilizer (500 kg / mu of commercial organic fertilizer; 27 kg / mu of ternary compound fertilizer (21-6-13)) and 0.5 kg / mu of microbial inoculant were mixed evenly, and then evenly applied on the surface of the crushed brassica napus tail meal, and the brassica napus tail meal and microbial inoculant were spun into the field with a rotary cultivator, and the straw was rotary cultivated to about 20 cm; the lower crop lettuce was planted on July 24.

[0178] (2) Detection of brassica napus tail meal decomposition rate

[0179] In order to clarify the effect of returning the tail meal of the open field vegetable to the field by using the microbial decomposition and returning technology on the decomposition rate of the tail meal, the nylon bag method was used to determine the decomposition rate. Four nylon bags were placed in each treatment, 40 g of straw was placed in each nylon bag, and the nylon bags were recovered on the 5th, 10th, 15th and 20th day after returning, the tail meal was washed and dried, and the decomposition rate was detected.

[0180] The calculation method of the tail meal decomposition rate is as follows:

[0181] Tail meal decomposition rate = (initial tail meal dry mass (g) - tail meal dry mass (g) at the end of incubation) / initial tail meal dry mass (g) x 100%.

[0182] Tail meal decomposition rate = (initial tail meal dry mass (g) - tail meal dry mass (g) at the end of incubation) / initial tail meal dry mass (g) x 100%.

[0183] The test results are shown in Table 6.

[0184] Table 6 Decomposition rate of brassica napus tail meal

[0185]

[0186]

[0187] As shown in Table 6, compared with the control group and the test group, the decomposition rate of broccoli tail leaves was measured by nylon mesh bag method, and the results showed that the decomposition rate of broccoli tail leaves in the treatment group was the highest, and the decomposition rate reached 98.98% after 20 days of treatment. Therefore, the microbial agent provided by the present application can effectively improve the decomposition rate of tail leaves in the technology of microbial decomposition and return of tail leaves of open field vegetables.

[0188] (3) Yield measurement of the following crop lettuce

[0189] After the broccoli tail leaves were returned to the field, the yield of the following lettuce was measured in September 2023. There were 3 groups in the experiment, 3 parallels in each group, a total of 9 treatments, and 1m 2 of sample was set for each treatment. The weight of lettuce leaves, roots, and net leaves, and the number of lettuce plants per 1m 2 were calculated to estimate the weight of tail leaves, the weight of net leaves, and the total weight in each treatment.

[0190] The test results are shown in Table 7 and Figure 7 .

[0191] Table 7 Yield of lettuce under different treatments

[0192]

[0193] As shown in Table 7, the growth of the following crop lettuce after the broccoli tail leaves were returned to the field was determined by yield index, and the results showed that: compared with the blank group, the control group and the test group, the net yield of lettuce in the test group was significantly higher than that in other treatments, reaching 7260.30 kg / mu, and the yield of lettuce in the blank group was the lowest, reaching 5779.56 kg / mu. The results showed that the microbial agent provided by the present application can significantly improve the yield of the following crop when used for microbial decomposition and return of tail leaves of open field vegetables.

[0194] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples without contradiction.

[0195] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the same; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A type of Bacillus subtilis ( Bacillus subtilis KF-002, characterized in that, The Bacillus subtilis KF-002 is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 33122.

2. A culture, characterized in that, The culture comprises the Bacillus subtilis KF-002 according to claim 1.

3. A fermentation broth, characterized in that, The fermentation liquor comprises the Bacillus subtilis KF-002 according to claim 1.

4. A bacterial suspension, characterized in that, The bacterial suspension comprises the Bacillus subtilis KF-002 according to claim 1.

5. A microbial inoculant, characterized in that, The microbial agent is composed of Bacillus pumilus (Bacillus subtilis) Bacillus pumilus ) KF-001 and Bacillus subtilis KF-002 according to claim 1. The Bacillus pumilus KF-001 is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 33121.

6. The microbial inoculant of claim 5, wherein, The effective viable cell number of the Bacillus subtilis KF-002 in the microbial inoculant is 6 x 10 9 -7 x 10 9 CFU / g.

7. The microbial inoculant of claim 6, wherein, The effective viable cell number of the Bacillus subtilis KF-002 in the microbial inoculant is 6.5 x 10 9 CFU / g.

8. The microbial inoculant of claim 5, wherein, The effective viable cell number of the Bacillus pumilus KF-001 in the microbial inoculant is 5 x 10 9 -6 x 10 9 CFU / g.

9. The microbial inoculant of claim 8, wherein, The effective viable cell number of the Bacillus pumilus KF-001 in the microbial inoculant is 5.4 x 10 9 CFU / g.

10. The use of the Bacillus subtilis KF-002 according to claim 1, the culture according to claim 2, the fermentation liquor according to claim 3, the bacterial suspension according to claim 4, or the microbial inoculant according to claim 5 in the field of fast composting of tailings.

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