Method for preparing organic fertilizer by using pomelo inferior fruits and application thereof

Abstract

The application discloses a method for preparing organic fertilizer by using pomelo residual and defective fruits and application thereof, and belongs to the technical field of agricultural waste resource utilization and microbial fertilizer. The method utilizes microbacterium A5, bacillus flexus HL-37 and bacillus cereus TH-35 to degrade pomelo residual and defective fruits, and prepares organic microbial fertilizer. Through a segmented aerobic fermentation process, the prepared liquid microbial fertilizer is mixed with soybean meal / fecal fertilizer at a ratio of 1:1 to be applied to a pomelo orchard, so that the weight of pomelo fruits is increased by 10.2%, the sugar content is increased by 5.3%, the odor is eliminated, and the alkaline soil pH value is neutralized. When the solid organic fertilizer is used for vegetable planting, the yield is increased by 23.8% compared with chemical fertilizer, and the soil microbial structure is significantly improved. The application realizes the resource utilization of agricultural wastes, and has the environmental protection and yield increasing benefits.

Description

Technical Field

[0001] This invention relates to the field of agricultural waste resource utilization and microbial fertilizer technology, and in particular to a method for preparing organic fertilizer from substandard pomelo fruit and its application. Background Technology

[0002] With the increasing scale of pomelo cultivation in major producing areas, the biomass of substandard fruit and fruit with no fresh consumption value generated annually through fruit thinning is enormous, twice that of fresh pomelo fruit. Because pomelo peels are thick and tough, they are difficult for natural microorganisms to degrade, and when piled up in fields, they become a new form of agricultural waste, creating a huge environmental burden. Therefore, how to quickly return substandard fruit resources to the fields and achieve resource reuse of agricultural waste is an urgent problem to be solved.

[0003] Organic fertilizer is a carbon-containing substance extracted from plants and animals and applied to the soil to provide nutrients for plant growth. Organic fertilizers, including compost, biogas slurry, and green manure, are residues extracted from fertilizers after a period of decomposition by plants and animals. In a narrower sense, organic fertilizer refers to substances that require specific processing to remove harmful substances. Based on their properties and functions, organic fertilizers can be categorized into organic fertilizers and urine fertilizers, straw fertilizers, green manures, soil fertilizers, and microbial fertilizers. The application of organic fertilizers in agricultural production not only improves yield and quality but also alters soil composition, properties, and microorganisms. Organic fertilizers can change the porosity of soil particles, forming a more suitable soil aggregate structure. Organic fertilizers also change soil composition, including elements such as nitrogen, phosphorus, and potassium, as well as organic matter that exists as soil nutrients, providing the soil with the nutrients needed for crop growth and accelerating crop development. Applying organic fertilizers can alter the amount and proportion of humic acid, increasing the amount of humic acid and activating its quality, thereby improving soil fertility. Organic fertilizers can not only improve soil quality but also promote crop growth and improve crop quality, thus contributing to agricultural development in many ways.

[0004] Pomelo fertilizer belongs to the category of green manure in organic fertilizers. This invention utilizes on-site, segmented aerobic fermentation of substandard pomelo fruit, using the fermentation products as organic fertilizer for vegetable and pomelo cultivation. Comparison of soil microbial community composition, soil physicochemical properties, and crop quality before and after the application of this organic fertilizer confirms the effectiveness of the green microbial fertilizer formed after rapid aerobic fermentation of pomelo peel and substandard fruit. Firstly, it solves the problem of high costs associated with the collection, transportation, and centralized processing of substandard pomelo fruit, achieving on-site return of agricultural waste to the field and resource recycling. Secondly, it provides a new type of organic fertilizer for vegetable and fruit cultivation, which is of great significance for the efficient utilization of biomass and the development of a biological circular economy, and for science and technology to support rural revitalization. Thirdly, the organic fertilizer derived from pomelo fruit degradation has a distinctive essential oil aroma unique to pomelo. When mixed with organic fertilizers such as poultry and livestock manure and peanut bran produced through anaerobic fermentation, it can effectively eliminate the odor produced by other organic fertilizers, acting as a deodorizer, promoting sustainable agricultural development, reducing negative environmental impacts, and driving the development of green agriculture. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing organic fertilizer from substandard pomelo fruit and its application, in order to solve the problems existing in the prior art. The organic fertilizer provided by this invention can be used in vegetable and pomelo cultivation, which can increase the weight of pomelo fruit by 10.2% and the sugar content by 5.3%, while eliminating odor. When solid organic fertilizer is used in vegetable cultivation, the yield is increased by 23.8% compared with chemical fertilizer, and the soil microbial structure is significantly improved.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for preparing organic fertilizer from substandard grapefruit fruit, comprising any one of the following steps:

[0008] Bacillus curvatureis HL-37 was inoculated into grapefruit culture medium No. 1 to obtain HL-37 fermentation broth;

[0009] Bacillus cereus TH-35 was inoculated into grapefruit culture medium No. 1 to obtain TH-35 fermentation broth;

[0010] Microbacterium A5 was inoculated into grapefruit culture medium No. 1 and cultured; then inoculated into grapefruit culture medium No. 2 and cultured; then inoculated into grapefruit culture medium No. 3 and cultured to obtain complete grapefruit fermentation broth of Microbacterium A5;

[0011] Add the HL-37 fermentation broth and the TH-35 fermentation broth to the Microbacterium A5 complete grapefruit fermentation broth, and continue fermentation to obtain liquid microbial fertilizer;

[0012] The liquid microbial fertilizer is dried to obtain solid organic fertilizer;

[0013] The No. 1 grapefruit culture medium: 250g of substandard grapefruit fruit was added to 500mL of LB medium, and water was added to bring the volume to 1L;

[0014] The No. 2 grapefruit culture medium: 400g of substandard grapefruit fruit were added to 200mL of LB medium, and water was added to bring the volume to 1L;

[0015] The No. 3 grapefruit culture medium consists of 500g of substandard grapefruit fruit, diluted with water to a final volume of 1L.

[0016] Optionally, the Bacillus curvatureis HL-37 has the accession number GDMCC No. 60045, the Bacillus cereus TH-35 has the accession number GDMCC No. 60044, and the Microbacterium A5 has the accession number CCTCC NO: M209174.

[0017] Optionally, the inoculum size of the microbacterium A5 is 5-15%.

[0018] Optionally, the inoculation amount of both the HL-37 fermentation broth and the TH-35 fermentation broth is 10%.

[0019] The present invention also provides a liquid microbial fertilizer prepared according to the method.

[0020] The present invention also provides a solid organic fertilizer prepared according to the method.

[0021] The present invention also provides the application of the liquid microbial fertilizer in promoting the growth of grapefruit.

[0022] Optionally, promoting grapefruit growth includes increasing the weight of individual grapefruits and / or their sugar content.

[0023] The present invention also provides the application of the solid organic fertilizer in promoting vegetable growth.

[0024] Optionally, promoting vegetable growth includes increasing the fresh weight and / or plant height of vegetables;

[0025] The vegetables mentioned include amaranth.

[0026] The present invention discloses the following technical effects:

[0027] This invention provides a method for preparing organic microbial fertilizer by degrading substandard grapefruit fruit using three bacterial strains (Microbacterium A5, HL-37, and TH-35). Through a segmented aerobic fermentation process, the prepared liquid microbial fertilizer is mixed with soybean meal / manure at a 1:1 ratio and returned to the grapefruit orchard. This increases grapefruit weight by 10.2% and sugar content by 5.3%, while simultaneously eliminating the odor from anaerobic fermentation of soybean meal / manure and neutralizing the pH of alkaline soil. When the solid organic fertilizer is used in vegetable cultivation, yields are increased by 23.8% compared to chemical fertilizers, and the soil microbial structure is significantly improved. This invention achieves the resource utilization of agricultural waste, combining environmental protection and increased yield benefits. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 The colony morphology of Microbacterium A5;

[0030] Figure 2 The growth curve during the propagation of Microbacterium A5 seeds;

[0031] Figure 3 The growth curve of Microbacterium A5 during amplified fermentation process;

[0032] Figure 4 Alpha diversity of rhizosphere soil microorganisms in different treatment groups;

[0033] Figure 5 PCA analysis chart;

[0034] Figure 6 For sample clustering heatmaps;

[0035] Figure 7 This is a map showing species distribution; the left side represents the phylum level, and the right side represents the class level.

[0036] Figure 8 A heatmap of species abundance clustering;

[0037] Figure 9 This is a network diagram of species at the genus level. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] The microbacterium A5 used in this invention, with accession number CCTCC NO: M209174, has been disclosed in patent CN101701198B, a strain producing gum-degrading enzyme and its application in the preparation of gum polysaccharide; Bacillus flexus HL-37, with accession number GDMCC No. 60045, has been disclosed in patent CN106591173A, a Bacillus flexus HL-37 that can activate heavy metal cadmium in soil and its application; Bacillus cereus TH-35, with accession number GDMCC No. 60044, has been disclosed in patent CN106167776A, a Bacillus cereus TH-35 that can activate heavy metal cadmium in soil and its application.

[0044] Example 1

[0045] 1. Preparation of grapefruit culture medium

[0046] Pre-treatment of substandard grapefruit: Wash the grapefruit with water to remove dirt and impurities from the peel. Cut into small pieces of 2-3cm.

[0047] (1) No. 1 grapefruit culture medium: 500 mL LB medium + 250 g grapefruit peel pieces per liter, add water to make up to 1 L, and adjust the pH to 4 with 0.2 mol / L NaOH. Dispense into 250 mL Erlenmeyer flasks, 150 mL / flask, sterilize at 121℃ for 20 min.

[0048] (2) No. 2 grapefruit culture medium: 200mL LB medium + 400g grapefruit peel pieces, add water to make up to 1L, adjust pH to 4 with 0.2mol / L HCl, dispense into 1L shake flasks, 500mL / flask, sterilize at 121℃ for 20min.

[0049] (3) No. 3 grapefruit culture medium: 500g grapefruit pieces, add water to make up to 1L, adjust pH to 4 with 1mol / L NaOH, sterilize at 121℃ for 20min.

[0050] 2. Domestication and scale-up fermentation of grapefruit peel degrading bacteria

[0051] (1) Streak the Microbacterium A5 strain stored at 4℃ on an LB solid medium plate and incubate at 30℃ for 24 hours. Pick a single colony and inoculate it into a 4mL LB liquid medium test tube for activation. Incubate at 30℃ and 150rpm / min for 12 hours.

[0052] (2) Take 3 mL of bacterial culture and inoculate it into 150 mL of No. 1 grapefruit culture medium. Incubate at 30℃ and 150 rpm for 3 days with shaking. Once the grapefruit particles in the 150 mL medium / 250 mL Erlenmeyer flask have largely degraded and no large particles remain, transfer 100 mL of this culture medium to a 500 mL No. 2 grapefruit culture medium / 1 L Erlenmeyer flask and incubate at 30℃ and 150 rpm for 5-7 days. During fermentation, take samples every 4 hours and measure the OD. 600 Finally, the growth curve of Microbe A5 was plotted. (Vertical axis: OD) 600 (x-axis: shaker fermentation time).

[0053] (3) After the grapefruit particles in the 1L conical flask disappear and become a paste, inoculate 1L of fermentation product into grapefruit culture medium No. 3 in a 10L or 15L plastic bucket. Cover the bucket with double gauze or 300 mesh gauze. Keep it at room temperature and open the gauze once a day to stir and ventilate until the grapefruit particles are completely turned into a paste, and obtain the complete grapefruit fermentation liquid of Microbacterium A5.

[0054] 3. Microbial strain combination

[0055] Three strains of bacteria, namely Microbacterium A5, Bacillus HL-37, and Bacillus cereus TH-35, were used for staged fermentation.

[0056] (1) Preparation of HL-37 fermentation broth for producing siderophores: 1 mL of frozen liquid seed culture was inoculated into 4 mL of LB medium and cultured with shaking for 12 hours, then inoculated into 100 mL of LB medium and cultured with shaking for 12 hours. Finally, 15% of the inoculum was inoculated into grapefruit culture medium No. 1 and cultured with shaking for 12 hours to obtain a viable count ≥10. 9 Fermentation broth at CFU / mL.

[0057] (2) Preparation of TH-35 fermentation broth for siderophore production: 1 mL of frozen liquid seed culture was inoculated into 4 mL of LB medium and cultured with shaking for 12 hours. Then, it was inoculated into 100 mL of LB medium and cultured with shaking for 12 hours. Finally, it was inoculated into No. 1 grapefruit culture medium at a 15% inoculation rate and cultured with shaking for 12 hours, yielding a viable count ≥10⁻⁶. 9 Fermentation broth at CFU / mL.

[0058] (3) Preparation of liquid / solid microbial fertilizer

[0059] After pretreatment of substandard pomelo fruit, the microbacterium A5 was acclimatized step by step through pomelo fruit culture medium No. 1 → pomelo fruit culture medium No. 2 → pomelo fruit culture medium No. 3. Then, it was mixed with HL-37 fermentation broth and TH-35 fermentation broth at a volume ratio of 10:1:1 to obtain liquid microbial fertilizer.

[0060] HL-37 fermentation broth / TH-35 fermentation broth are mixed with Microbacterium A5 complete grapefruit fermentation broth at a volume ratio of 1:10 to obtain a two-strain combined liquid microbial fertilizer.

[0061] Liquid microbial fertilizer is dried at 60℃ for 5 hours to obtain liquid organic fertilizer.

[0062] 4. Field-scale microbial fertilizer preparation process

[0063] Disinfect a 200L plastic bucket with diluted 84 disinfectant. Add 5cm pieces of damaged young fruit (weighing 75kg), filling the bucket to two-thirds full. Add 75L of water to submerge the fruit pieces, bringing the total volume to 150L. Add 10-15L of Microbacterium A5 complete grapefruit fermentation liquid, stir well, and cover the bucket with a 200-mesh screen. Stir once a day. After 3-4 days, the fruit pieces will completely become a paste, which can be used as a field fermentation medium. Inoculate with the prepared HL-37 and TH-35 fermentation liquids at a 10% volume ratio, continuing to stir once a day. After 2 days, a liquid microbial fertilizer will be ready for use.

[0064] Solid organic fertilizer was prepared by drying liquid microbial fertilizer at 60℃ for 5 hours.

[0065] Application method: Liquid microbial fertilizer for pomelo orchards: Mix with anaerobic fermented organic fertilizers such as soybean meal / manure (the volume ratio of soybean meal to manure is 1.5:4.5) at a volume ratio of 1:1. Apply 20 catties per tree, once every two weeks, for a total of four times.

[0066] Solid organic fertilizer is used in vegetable cultivation: it replaces chemical fertilizer, and the application rate is 25g / kg of soil (200mL / kg for liquid fertilizer).

[0067] 5. Results

[0068] 5.1 Colony morphology of Microbacterium A5

[0069] like Figure 1 As shown, the starting colonies of Microbacterium A5 used for grapefruit peel degradation are round, pale yellow, thick in the middle and thin at the edges, and relatively moist.

[0070] 5.2 Acclimation of the degradation activity of microbes on grapefruit peel and growth curves during scale-up fermentation

[0071] The scale-up process of microbial fermentation for grapefruit degradation involves two aspects: firstly, gradually enhancing the degradation activity of the microbial strain on grapefruit peel; and secondly, increasing the processing capacity and shortening the fermentation cycle. After this scale-up process, as... Figure 2 As shown, under the conditions of 30℃ and 150rpm shaking culture, the microbes entered the logarithmic growth phase after 30-36 hours. The fermentation broth at the mid-to-late logarithmic growth stage (35-36 hours) was determined to be the seed culture for the next step of scale-up fermentation (500mL / 1L). Figure 3 As shown, in a medium containing 80% grapefruit, and with shaking at 30°C and 150 rpm / min, the microbes entered the logarithmic growth phase after 20-28 hours. The fermentation broth after 24 hours was determined to be the seed culture inoculated into the complete grapefruit peel medium.

[0072] 5.3 Degradation rate of substandard grapefruit fruit

[0073] The degradation efficiency of substandard pomelo fruit by the three-strain synergistic fermentation and single / two-strain combinations is shown in Table 1. The degradation rate of the main components of pomelo peel (cellulose, hemicellulose and lignin) by the three-strain synergistic fermentation compound agent is shown in Table 2.

[0074] Table 1 Comparison of degradation efficiency of substandard grapefruit fruit by synergistic fermentation of three strains and single / two strain combinations

[0075]

[0076] Table 2. Degradation rate of main components of pomelo peel by the three-strain synergistic fermentation compound inoculant.

[0077] Element Initial content Content after fermentation Degradation rate Cellulose 32.5％ 2.6％ 92.0％ hemicellulose 28.7％ 3.4％ 88.1％ Lignin 18.3％ 6.4％ 65.0％

[0078] Example 2: Application of liquid microbial fertilizer in a pomelo orchard

[0079] 1. Materials and Methods

[0080] The liquid microbial fertilizer prepared using the field-scale microbial fertilizer preparation process described in Example 1, Section 4 was used in the experiment.

[0081] Four treatment groups (n=20 trees) were set up in Meizhou Songyuan Pomelo Orchard: T1: conventional fertilizer; T2: liquid microbial fertilizer + soybean meal / manure (the volume ratio of liquid microbial fertilizer to soybean meal / manure was 1:1, and the volume ratio of soybean meal to manure was 1.5:4.5); T3: soybean meal / manure (anaerobic composting); T4: blank control.

[0082] Fertilization plan: For group T2, apply 20 catties of mixed fertilizer (10 catties of liquid microbial fertilizer + 10 catties of soybean meal / manure) to each tree each time, for a total of 4 applications, with an interval of 14 days; other groups should apply the corresponding fertilizer according to the same treatment.

[0083] Testing indicators: When the pomelo fruit is ripe, the weight of a single fruit and its sugar content (using a handheld saccharimeter) are measured. Soil samples are also collected to test organic matter and alkaline nitrogen content. Specific methods for testing the concentration of volatile organic compounds (VOCs) in the air, sensory evaluation criteria for odor, and soil element and organic matter content are as follows.

[0084] 1.1 Soil element and organic matter content detection

[0085] (1) Soil organic matter content detection

[0086] The potassium dichromate oxidation-external heating method (classical method) was used.

[0087] Standard basis: Soil testing - Part 6: Determination of soil organic matter (NY / T 1121.6-2006).

[0088] Testing Procedure: Weigh an appropriate amount of air-dried soil sample, add excess potassium dichromate-sulfuric acid solution, and heat in an oil bath to oxidize the organic matter. Titrate the remaining potassium dichromate with ferrous sulfate solution, and calculate the organic matter content (converted to carbon content, unit: g / kg) based on the amount consumed.

[0089] (2) Soil element detection

[0090] Pretreatment method: Soil digestion: Soil is digested with a mixture of hydrofluoric acid, nitric acid and perchloric acid to convert elements into ionic states (standard such as HJ 803-2016).

[0091] Elemental determination: Inductively coupled plasma optical emission spectrometry (ICP-OES), standard: HJ 700-2014.

[0092] 1.2 Determination of volatile organic compound (VOCs) concentration in air

[0093] The determination was performed using a portable PID analyzer.

[0094] a. Instrument preparation and calibration

[0095] Power-on warm-up: Connect the power supply and allow the instrument to warm up for 10-15 minutes until the indicator lights stabilize (to avoid drastic changes in ambient temperature affecting accuracy).

[0096] Zero-point calibration: Pass clean, odorless air (or high-purity nitrogen) through the sampling tube into the instrument for 1-2 minutes, and adjust the instrument display to 0 ppm (or perform automatic zero-point calibration according to the instruction manual). Span calibration (optional): Connect isobutylene standard gas (known concentration) and pass it into the instrument at a flow rate of 100-200 mL / min. After the reading stabilizes, adjust the instrument to match the standard gas concentration (calibration frequency: before daily testing or after instrument malfunction).

[0097] b. On-site sampling and testing

[0098] Direct sampling: Place the sampling probe at the detection point (about 1.5m above the ground), turn on the instrument's pump function, and directly draw in the air sample (the sampling flow rate is usually 200-500mL / min).

[0099] Adsorption tube sampling (if enrichment is required): After sampling with activated carbon or Tenax tubes, the sample is introduced into the PID detector by thermal desorption or solvent desorption (suitable for low concentration scenarios).

[0100] c. Detection procedure:

[0101] Insert the sampling probe into the environment to be tested, avoiding contact with water or particulate matter (install a filter head if necessary).

[0102] Start the detection program, and the instrument will display the VOCs concentration value in real time (usually updated once per second), and record the highest value or the average value after stabilization.

[0103] If multiple sampling points are being tested, the sampling tube should be purged with clean air for 1-2 minutes when switching points to avoid cross-contamination.

[0104] d. Data recording and processing

[0105] Real-time recording: Synchronously records the detection time, location, ambient temperature (PID response is affected by temperature and needs correction), humidity (humidity > 80% may affect accuracy, dehumidification is recommended), and the concentration value displayed by the instrument.

[0106] Unit conversion: Convert ppm to mg / m³ using the formula. 3 Concentration (mg / m³) 3 = Concentration (ppm) * Molecular weight / 24.45;

[0107] Note: 24.45 is the molar volume of gas under standard conditions, in L / mol; the molecular weight is the average molecular weight of the VOCs mixture, or assumed to be the molecular weight of isobutylene (56.1).

[0108] 1.3 Odor Sensory Scoring Criteria (Three-Point Comparison Odor Bag Method)

[0109] Standard basis: "Determination of malodor in air quality - Three-point comparison odor bag method" (GB / T 14675-1993).

[0110] Requirements for olfactory assessors: Train 6 olfactory assessors (without olfactory impairment), aged 18-45.

[0111] Sample preparation: The sample gas was diluted stepwise with odorless air to prepare gas bags with different dilution ratios.

[0112] Odor identification procedure: Each time, provide the odor tester with 3 gas bags, one containing sample dilution gas and two containing odorless air, and ask them to identify the bag with an odor. Test step by step from low concentration to high concentration until the odor tester can no longer identify it correctly, and record the lowest dilution factor at which the odor can be detected (odor threshold).

[0113] The scoring criteria are shown in Table 3.

[0114] Table 3 Odor Sensory Scoring Criteria

[0115]

[0116] 2. Experimental Results

[0117] 2.1 Effects of liquid microbial fertilizer on average weight of single grapefruit, sugar content, soil organic matter, and available nitrogen content.

[0118] The average weight of a single pomelo, sugar content, soil organic matter, and available nitrogen content for each group are shown in Table 4.

[0119] Table 4. Average weight of single pomelo fruit, sugar content, soil organic matter, and available nitrogen content for each group.

[0120] Group Average weight of a single fruit / g Sugar content / % Soil organic matter / % Alkaline nitrogen / mg / kg T1 856±32 12.5±0.8 1.82±0.15 156.7±9.2 T2 943±28 17.8±0.5 3.14±0.12 189.3±11.5 T3 835±25 12.7±0.6 1.79±0.13 152.4±8.7 T4 720±20 10.2±0.4 1.35±0.10 112.5±7.3

[0121] Note: Compared with the T4 control group without fertilizer, the application of chemical fertilizer and soybean meal / manure can increase the weight of pomelo fruit, but the T2: liquid microbial fertilizer + soybean meal / manure (1:1) showed the most significant increase in average weight of single fruit, sugar content, soil organic matter and alkaline nitrogen.

[0122] 2.2 Deodorizing effect

[0123] After fertilization, the concentration of volatile organic compounds (VOCs) in the orchard air decreased by 68.5% compared with that of the T3 group, and the odor sensory score dropped from 5.2 points (strong odor) to 1.8 points (no obvious odor).

[0124] 2.3 Effects of liquid microbial fertilizer on soil element and organic matter content

[0125] Table 5 Soil element and organic matter content for each group

[0126]

[0127]

[0128] As shown in Table 5, (1) Organic matter and soil fertility were significantly improved. After applying pomelo peel organic fertilizer, the soil organic matter content soared from 0.19% to 17.46%, an increase of 81.4 times, indicating that the soil structure and fertilizer retention capacity were significantly improved; at the same time, the electrical conductivity increased from 57 μS / cm to 983.1 μS / cm, indicating that the content of soluble nutrients (such as nitrogen, phosphorus and potassium ions) in the soil increased significantly, providing more sufficient nutrition for plant growth. (2) The content of nitrogen, phosphorus and potassium elements increased significantly. Total nitrogen content increased from 205.17 mg / kg to 6247.21 mg / kg, and available nitrogen (nitrogen forms that plants can directly absorb) increased from 24.42 mg / kg to 177.5 mg / kg, an increase of 6.3 times, promoting plant leaf growth and photosynthesis; available phosphorus content increased from 75.72 mg / kg to 1945.09 mg / kg, an increase of 24.4 times, significantly enhancing plant root development and fruit quality; available potassium increased from 64.00 mg / kg to 2185.85 mg / kg, an increase of 33.8 times, contributing to plant stress resistance (such as drought resistance and cold resistance) and fruit sugar accumulation. (3) Calcium, magnesium, iron and other micronutrients were synergistically improved. The contents of micronutrients such as calcium, magnesium, and iron all increased significantly (e.g., iron content increased by 122.7 times), which can prevent plant nutrient deficiencies and promote fruit development; the copper content decreased slightly, which may be related to the slow-release characteristics of copper in organic fertilizer, but overall it is still within the range suitable for plant absorption. (4) Adaptive adjustment of pH value. The soil pH value decreased from 6.76 (slightly alkaline) to 4.25 (acidic), which is close to the pH value of the organic fertilizer itself (4.09). Although the acidity increased, crops such as pomelo are suitable for slightly acidic soil (pH 5.5-6.5), and the increase in organic matter can buffer the changes in soil acidity and alkalinity. In actual use, it did not inhibit plant growth.

[0129] Meanwhile, the liquid microbial fertilizer prepared by this invention was compared with single-strain fermentation (liquid microbial fertilizer for complete grapefruit degradation prepared only with A5 microbacterium) and natural composting (natural composting by burying defective grapefruit in the soil). The results are shown in Table 6.

[0130] Table 6 Comparison of Liquid Microbial Fertilizer with Existing Technologies

[0131] index This invention Single-strain fermentation Natural composting Degradation cycle 3-7 days (until completely pasty) 10 days 90-180 days Grapefruit degradation rate 95.3% (weight loss rate) 62.7％ <30％ Odor removal rate 68.5% (VOCs concentration decreased) <30％ none Increased weight of individual pomelo fruits 10.2% (20 catties / tree, 4 applications) <5％ none Soil organic matter increase factor 81.4 times (from 0.19% to 17.46%) <10 times <5x Nitrogen, phosphorus and potassium retention rates Total nitrogen retention rate: 82.3% Total nitrogen retention rate: 58.6% Total nitrogen loss exceeds 40%

[0132] In summary, this embodiment verifies that the application of liquid microbial fertilizer significantly increases the content of organic matter and key nutrients such as nitrogen, phosphorus, and potassium in the soil, while also improving the supply of micronutrients, creating a better soil environment for plant growth. Data shows that this organic fertilizer is highly effective in improving soil fertility and promoting nutrient balance, significantly outperforming existing technologies, and has the effect of promoting growth and improving soil quality.

[0133] Example 3: Application of solid organic fertilizer in vegetable (amaranth) cultivation

[0134] 1. Experimental Design and Treatment Group Setup

[0135] The settings for each processing group are shown in Table 7.

[0136] Compound fertilizer: Purchase vegetable-specific compound fertilizer (Stanley, SDL-FHF01) from the market.

[0137] Table 7. Settings for each treatment group

[0138] Processing group Fertilizer type Application rate Soil type Group T1 (blank control) No fertilizer - Black soil, red soil, loess Group T2 Liquid grapefruit peel microbial fertilizer 200 mL / kg soil Black soil, red soil, loess Group T3 Solid grapefruit peel microbial fertilizer 25g / kg soil Black soil, red soil, loess Group T4 (Commercial Compound Fertilizer) Compound fertilizer 2.5g / kg soil Black soil, red soil, loess

[0139] 2. Results

[0140] 2.1 Effects of Soil Physicochemical Properties on Amaranth Growth Physical Indicators

[0141] Table 8 shows the comparison of physical indicators of amaranth growth in each treatment group (60 days after planting), and Table 9 shows the changes in soil physicochemical properties (60 days after planting).

[0142] Table 8 shows that the fresh weight of GSF was significantly higher than that of Con: the fresh weight of GSF in black soil was 3.8 times that of Con, while in red soil and loess it was 3.2 times and 2.9 times, respectively. Furthermore, the fresh weight of GSF was superior to that of GLF: the fresh weight of GSF in black soil was 15.3% higher than that of GLF, while in red soil and loess it was 12.1% and 9.7% higher, respectively. CF had the best effect on fresh weight: the fresh weight of CF in black soil was 22.4% higher than that of GSF, but the difference between GSF and CF narrowed in red soil and loess (10.8% and 8.3% lower, respectively). GSF significantly promoted plant height growth: the plant height of GSF in black soil was 3.1 times that of Con, while in red soil and loess it was 2.9 times and 2.7 times, respectively. Furthermore, the plant height of GSF was superior to that of GLF: the plant height of GSF in black soil was 18.7% higher than that of GLF, while in red soil and loess it was 14.3% and 11.3% higher, respectively. CF plants were the tallest: in black soil, CF plants were 25.2% taller than GSF plants, but GSF plants were less taller than CF plants in red soil and loess soil (11.6% and 9.1% shorter, respectively).

[0143] Table 9 shows that GSF significantly increased soil organic matter: in black soil, GSF organic matter was 2.1 times higher than Con, and in red soil and loess it was 2.3 times and 2.6 times higher, respectively. GSF's organic matter content was superior to GLF: in black soil, GSF organic matter was 19.5% higher than GLF, and in red soil and loess it was 18.0% and 17.9% higher, respectively. CF had a limited effect on increasing organic matter: in black soil, CF organic matter was close to that of GSF (only 5.4% higher), while in red soil and loess it was lower than that of GSF (12.6% and 15.3% lower, respectively). GSF lowered soil pH: in black soil, GSF pH was 2.3 lower than Con, and in red soil and loess it was 1.8 and 1.5 lower, respectively, possibly related to the acidity of organic fertilizer (pH 4.09). GSF had a more significant effect on pH: the pH of GSF in black soil was 0.4 lower than that of GLF, while it was 0.3 and 0.2 lower in red soil and loess soil, respectively, but it did not completely inhibit amaranth growth (because the initial pH of black soil was closer to neutral). CF had a smaller effect on pH: the pH of CF in black soil was 0.8 higher than that of GSF, while it was 0.6 and 0.5 higher in red soil and loess soil, respectively.

[0144] Table 8 Comparison of physical indicators of amaranth growth in different treatment groups

[0145]

[0146]

[0147] Note: *p<0.05 compared to Con.

[0148] Table 9 Changes in soil physicochemical properties

[0149]

[0150] Note: *p<0.05 compared to Con.

[0151] In summary, solid organic fertilizer significantly promoted amaranth growth in all three soil types, increasing fresh weight and plant height by 2.7-3.8 times compared to the control group, and its effect was superior to that of liquid microbial fertilizer. In black soil, the growth indicators of GSF (such as fresh weight 33.9 g / plant and plant height 30.1 cm) were close to those of compound fertilizer (CF), but the gap with CF narrowed in red soil and loess soil, indicating its adaptability to different soil types.

[0152] After applying GSF, the organic matter content of the three soil types increased significantly (2.57% for black soil, 1.51% for red soil, and 1.24% for loess), which was 1.6-2.6 times higher than that of the control group and better than that of liquid fertilizer, indicating that its soil improvement effect is long-lasting.

[0153] Potential for partial replacement of chemical fertilizers: Although GSF has a slightly lower growth-promoting effect than CF, it is significantly better than CF in improving soil organic matter, and the growth gap between GSF and CF is small in red and loess soils, so it can be used as a chemical fertilizer alternative.

[0154] Acidity effects and adaptability: GSF application leads to a decrease in soil pH, but the effect is less in black soil (initial pH 6.64), where amaranth grows well; in red and loess soils, attention should be paid to acid-adjusting measures such as lime application to optimize the effect.

[0155] 2.2 Analysis of the impact on soil microorganisms

[0156] 2.2.1 Alpha Diversity

[0157] The alpha diversity of rhizosphere soil bacteria in amaranth was detected in the control group and the solid organic fertilizer group. The results were shown in Table 10 and Figure 4 Solid fertilizer treatment significantly reduced rhizosphere bacterial abundance, decreasing by 18.0% compared to the control group, possibly related to the acidity (pH 4.09) of organic fertilizer inhibiting some bacterial communities. Bacterial species richness decreased by 12.6%, indicating that solid fertilizer may have altered the soil microenvironment, leading to the disappearance of some species. Bacterial diversity decreased by 7.0%, suggesting a decrease in community structure complexity, but dominant bacterial communities may have become more concentrated. Species evenness decreased, with the dominance of dominant phyla (such as Proteobacteria) increasing under solid fertilizer treatment.

[0158] Table 10. Alpha diversity of amaranth rhizosphere soil bacteria in different treatment groups

[0159] index Blank group (Con) Solid fertilizer group (GSF) Significance of difference Remark ACE Index 2100±85 1722±68 p<0.0001 Abundance Chao1 Index 1950±72 1705±59 p<0.0001 Species richness Shannon Index 10.2±0.3 9.5±0.2 p<0.01 diversity Simpson index 0.985±0.004 0.972±0.003 p<0.1 Species evenness

[0160] 2.2.2 Beta Diversity

[0161] Beta diversity represents the abundance diversity of microorganisms among different samples. The beta diversity results of amaranth rhizosphere soil under different treatments are shown in the figure. (The figure is derived from PCA analysis.) Figure 5 As can be seen from the Venn diagram, the rhizosphere soil beta diversity under the four treatments is quite different and there is no overlap. The number of OTUs specific to the rhizosphere soil in Group 1, Group 2, Group 3 and Group 4 are 2572, 2513, 1722 and 1818 respectively. The number of OTUs shared by the four groups is 318, which is a low proportion, indicating that the community structure similarity among different samples is weak.

[0162] Sample clustering heatmap Figure 6 The GSF group and the liquid fertilizer group (GLF) clustered together, while the Con group and the CF group clustered together, indicating that the microbial community structure of the organic fertilizer treatment was more similar and significantly different from that of the compound fertilizer / blank group.

[0163] 2.2.3 Changes in dominant bacterial communities at the phylum / class level

[0164] The dominant bacterial phylum / class at the rhizosphere soil level of amaranth in the control group and the solid organic fertilizer group were detected, and the results were found (Table 11 and...). Figure 7 At the phylum level, the top 10 dominant bacterial groups in terms of relative abundance all reached over 90% in the 12 samples. Furthermore, the abundance of the top 10 dominant bacterial groups in the rhizosphere soil treated with the three fertilizers was higher than that in the control group. The main dominant phyla in the rhizosphere soil were Proteobacteria, Acidobabacteriota, Gemmatimonadota, and Firmicutes. At the phylum level, compared with the control group (Con), the application of compound fertilizer (CF) increased the abundance of Proteobacteria and Gemmatimonadota, and decreased the abundance of Gemmatimonadota and Firmicutes; the application of liquid organic fertilizer (GLF) increased the abundance of Proteobacteria and decreased the abundance of Gemmatimonadota; and the application of solid organic fertilizer (GSF) increased the abundance of Proteobacteria and decreased the abundance of Gemmatimonadota, Gemmatimonadota, and Firmicutes. In addition, all fertilizer-treated groups significantly reduced the abundance of Methylomirabilota. At the class level, the top 10 most abundant bacterial groups reached 72.0%, 75.0%, 77.0%, and 79% in the four treatments, respectively. In the rhizosphere soil, the dominant classes were Alphaproteobacteria, Gammaproteobacteria, Vicinamibacteria, and Gemmatimonadetes. After soil treatment, the relative abundance of Alphaproteobacteria and Gemmatimonadeta decreased, while the relative abundance of Bacilli increased significantly.

[0165] Table 11 Dominant bacterial groups at the phylum / class level

[0166]

[0167]

[0168] 2.2.4 Species and Network Analysis at the Genus Level

[0169] Species abundance clustering heatmap Figure 8 The highest abundance of genera in the Halanaerobiaeota phylum was observed in the GSF group, which may be related to the anaerobic metabolism of recalcitrant carbon sources in organic fertilizers; while the Deferrisomatota phylum was more abundant in the control group, indicating that solid fertilizers altered the microbial community related to carbon cycling.

[0170] Correlation network analysis ( Figure 9The abundance of Sphingomonas and Bacillus was high in the GSF group, and it was positively correlated with other genera (mainly red lines), suggesting that these bacteria may form a synergistic metabolic network under organic fertilizer treatment, promoting nutrient conversion.

[0171] In summary, solid organic fertilizer has a dual impact on microbial diversity: reduced diversity but enriched functional microbial communities. Solid fertilizer inhibits nutrient-tolerant microbial communities such as Acidobacteria and Bacillus, but promotes microbial communities in Proteobacteria and Firmicutes that are related to nutrient decomposition (such as nitrogen and phosphorus conversion) and plant growth promotion (such as Bacillus), which may optimize microbial community function through a "screening effect".

[0172] Potential effects of soil acidity: The application of solid fertilizers can cause the soil pH to drop to 3.54-4.34, which may inhibit neutral / alkaline bacteria, but enrich acidophilic bacteria (such as certain genera in the Proteobacteria phylum). Long-term use requires attention to acid-base balance.

[0173] Differences from compound fertilizer: The decline in microbial diversity under compound fertilizer treatment is less than that under solid fertilizer, but solid fertilizer may provide carbon source for specific functional bacteria by increasing organic matter (such as GSF in the previous table, which increased the organic matter content of black soil to 2.57%), which is beneficial to the long-term maintenance of soil fertility.

[0174] Example 4: Field Cultivation Experiment of Amaranth

[0175] 1. Field Trial Design

[0176] The seeds used are all-red amaranth produced by Nanjing Jia Hua Agricultural Development Co., Ltd.

[0177] Treatment groups: T1 (25g / kg of solid grapefruit peel fermented organic fertilizer), T2 (2.5g / kg of chemical fertilizer, commercially available vegetable-specific chemical fertilizer (Kunningwang, fhf)), T3 (blank). Each group was replicated 3 times, with a planting area of ​​4m×1.5m.

[0178] Field planting method: Apply the root fertilizer once, and carry out other field management measures in the conventional way, and water regularly in the morning and evening.

[0179] 2. Detection indicators

[0180] The planting cycle lasted a total of 65 days. After 65 days, the fresh weight, dry weight, plant height, and soil microbial diversity of amaranth were measured (16S rRNA sequencing).

[0181] 3. Results

[0182] 3.1 Effects on growth

[0183] The fresh weight, dry weight, and plant height of each group are shown in Table 12.

[0184] Table 12 Growth Indicators for Each Group

[0185] Processing group Fresh weight (g / plant) Dry weight (g / plant) Plant height (cm) Yield (kg / mu) T1 141±8*** 42±3*** 62±4*** 2840±120** T2 122±6* 35±2* 53±3* 2300±90* T3 26±2 12±1 11±1 520±30

[0186] Note: Compared with group T3, *p<0.05, ***p<0.01.

[0187] 3.2 Impact on soil microorganisms

[0188] Alpha diversity: The ACE index of soil bacteria in group T1 increased by 15.7% and the Shannon index increased by 12.3% compared with group T2;

[0189] Dominant microbial communities: The abundance of Bacillus in the rhizosphere soil of group T1 increased by 32.6% compared with group T2, while the abundance of Acidobacteriota decreased by 18.4%, indicating improved soil fertility.

[0190] 3.3 Effects on the KEGG pathway

[0191] The changes in KEGG pathway abundance are shown in Table 13.

[0192] Table 13. Changes in KEGG pathway abundance

[0193]

[0194]

[0195] Based on the above experimental results, this invention provides a method for preparing organic microbial fertilizer by degrading substandard pomelo fruit using three bacterial strains (Microbacterium A5, HL-37, and TH-35). Through a segmented aerobic fermentation process, the prepared liquid microbial fertilizer is mixed with soybean meal / manure at a 1:1 ratio and returned to the pomelo orchard. This increases the weight of the pomelo by 10.2% and the sugar content by 5.3%, while eliminating odor. When the solid organic fertilizer is used in vegetable cultivation, the yield is increased by 23.8% compared to chemical fertilizers, and the soil microbial structure is significantly improved. This invention achieves the resource utilization of agricultural waste, combining environmental protection and increased yield benefits.

[0196] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing organic fertilizer using substandard grapefruit fruit, characterized in that, Includes the following steps: Bacillus curvatureis HL-37 was inoculated into grapefruit culture medium No. 1 to obtain HL-37 fermentation broth; Bacillus cereus TH-35 was inoculated into grapefruit culture medium No. 1 to obtain TH-35 fermentation broth; Microbacterium A5 was inoculated into grapefruit culture medium No. 1 and cultured; then inoculated into grapefruit culture medium No. 2 and cultured; then inoculated into grapefruit culture medium No. 3 and cultured to obtain complete grapefruit fermentation broth of Microbacterium A5; Add the HL-37 fermentation broth and the TH-35 fermentation broth to the Microbacterium A5 complete grapefruit fermentation broth, and continue fermentation to obtain liquid microbial fertilizer; The No. 1 grapefruit culture medium: 250 g of substandard grapefruit fruit was added to 500 mL of LB medium, water was added to make up to 1 L, pH was adjusted to 4, and sterilized at 121℃ for 20 min. The No. 2 grapefruit culture medium: 200 mL of LB medium was added to 400 g of substandard grapefruit fruit, water was added to make up to 1 L, pH was adjusted to 4, and sterilized at 121℃ for 20 min; The No. 3 grapefruit culture medium: 500 g of substandard grapefruit fruit, add water to make up to 1L, adjust pH=4, sterilize at 121℃ for 20 min; The volume ratio of the Microbacterium A5 complete grapefruit fermentation broth, the HL-37 fermentation broth, and the TH-35 fermentation broth is 10:1:

1. The Bacillus curvatureis HL-37 has the accession number GDMCC No. 60045, the Bacillus cereus TH-35 has the accession number GDMCC No. 60044, and the Microbacterium A5 has the accession number CCTCC NO: M209174.

2. A liquid microbial fertilizer prepared according to the method of claim 1.

3. A solid organic fertilizer prepared according to claim 1, characterized in that, Liquid microbial fertilizer is prepared by the method according to claim 1, and the liquid microbial fertilizer is dried at 60°C for 5 hours to obtain solid organic fertilizer.

4. The application of the liquid microbial fertilizer as described in claim 2 in promoting the growth of grapefruit.

5. The application as described in claim 4, characterized in that, Promoting grapefruit growth includes increasing the weight of individual grapefruits and / or their sugar content.

6. The application of the solid organic fertilizer as described in claim 3 in promoting vegetable growth.

7. The application as described in claim 6, characterized in that, The promotion of vegetable growth includes increasing the fresh weight and / or plant height of vegetables; The vegetables mentioned include amaranth.

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