Method for improving quality of pig manure compost by using specific microbial inoculum combination

Abstract

The invention relates to the technical field of agricultural waste treatment, and discloses a method for improving pig manure compost quality by using a specific microbial inoculum combination, and the method comprises the following steps: S1, compost raw material preparation: selecting fresh pig manure and rice hull straw as compost raw materials; s2, microbial inoculum preparation: a specific microbial inoculum combination is prepared, and the specific microbial inoculum combination comprises bacillus licheniformis, bacillus megatherium, aspergillus niger, bacillus subtilis and bacillus cereus; s3, a composting process: uniformly mixing the fresh pig manure, the rice husk straws and the specific microbial inoculum combination, and composting by adopting a high-temperature aerobic composting method; and S4, monitoring, regulating and controlling: in the composting process, monitoring the temperature of a pile body every day, and turning the pile at regular intervals to maintain an aerobic condition. The passivation effect on heavy metals (such as Cu and Zn) in the composting process is remarkably improved through a specific fungicide combination, so that the bioavailability of the heavy metals is reduced, and the environmental risk when a compost product is applied to soil is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural waste treatment, and specifically to a method for improving the quality of pig manure compost using a specific combination of microbial agents. Background Art

[0002] In agricultural production, pig manure, as a common organic waste, has a large output and is usually converted into organic fertilizer through composting treatment for resource utilization. The existing pig manure composting technologies mainly include natural composting and aerobic composting. Among them, aerobic composting is widely used due to its high treatment efficiency and low environmental pollution risk. To further improve the composting effect, a variety of commercial microbial agents have been developed on the market. These microbial agents usually contain microorganisms such as Bacillus subtilis, Saccharomyces cerevisiae or Trichoderma viride, which can promote the composting process by accelerating the decomposition of organic matter and improve the quality of compost products to a certain extent.

[0003] However, there are still some deficiencies in the existing pig manure composting technologies. The cycles of natural composting and traditional aerobic composting are relatively long, usually taking 45 - 60 days to complete composting, and the degree of composting is not high, resulting in residual undecomposed organic matter in the compost products, which affects their applicability as fertilizers. In addition, the losses of nutrients such as nitrogen, phosphorus and potassium during the composting process are relatively serious. For example, nitrogen is often volatilized in the form of ammonia, and phosphorus and potassium may be reduced due to leachate loss, resulting in a decrease in the nutrient content of the compost. At the same time, heavy metals (such as Cu, Zn) commonly contained in pig manure are difficult to effectively passivate in the existing technologies, and the ability of commercial microbial agents to control heavy metals is limited, so there is a certain pollution risk when the compost products are applied to the soil. Summary of the Invention

[0004] Aiming at the deficiencies of the existing technology, the present invention provides a method for improving the quality of pig manure compost using a specific combination of microbial agents, which solves the problem that heavy metals (such as Cu, Zn) commonly contained in pig manure are difficult to effectively passivate in the existing technologies, and there is a certain pollution risk when the compost products are applied to the soil.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions: A method for improving the quality of pig manure compost using a specific combination of microbial agents, comprising the following steps: S1. Preparation of compost raw materials: Select fresh pig manure and rice husk straw as compost raw materials. Among them, fresh pig manure provides organic matter and nutrients, and rice husk straw is used to adjust the air permeability and carbon-nitrogen ratio of the compost; S2. Preparation of microbial agents: Prepare a specific combination of microbial agents, and the specific combination of microbial agents includes Bacillus licheniformis, Bacillus megaterium, Aspergillus niger, Bacillus subtilis and Bacillus cereus; S3. Composting process: Mix the fresh pig manure, rice husk straw and the specific combination of microbial agents evenly, and use the high-temperature aerobic composting method for composting; S4. Monitoring and regulation: During the composting process, the temperature of the compost pile is monitored daily, and the pile is regularly turned to maintain aerobic conditions.

[0006] Preferably, in the specific microbial agent combination, the strain ratio of Bacillus licheniformis, Bacillus megaterium, Aspergillus niger, Bacillus subtilis, and Bacillus cereus is 3:3:3:3:1 or 3:3:2:2:1.

[0007] Preferably, in step S1, the fresh pig manure is from a pig farm, and the rice husk straw is crushed to a particle size of 1 - 5 cm.

[0008] Preferably, in step S3, in the high-temperature aerobic composting method, the height of the compost pile is controlled at 1.5 - 2 m, and the bottom diameter is controlled at 2 - 3 m to promote heat accumulation and air circulation.

[0009] Preferably, in step S3, the composting period is 30 - 35 days, and the compost pile is set in a conical shape.

[0010] Preferably, in step S4, the monitoring and regulation include: S401. Measuring the temperature at five positions (east, south, west, north, and middle) of the compost pile daily using an alcohol thermometer, with an insertion depth of 20 - 30 cm, and recording the ambient temperature; S402. On the 1st, 6th, 11th, 16th, 20th, 23rd, 29th, and 34th days of composting, taking 30 g samples from each of the five positions of the compost pile, mixing them, and storing them under -20°C conditions.

[0011] Preferably, it further includes step S5. Physicochemical index determination, specifically: S501. Determination of pH value, conductivity, and E4 / E6 value: Taking 10.0 g of the compost sample and mixing it with 100 mL of distilled water at a mass ratio of 1:10, shaking it at 160 r / min for 1 hour and then filtering, and measuring the pH value, conductivity, and the absorbance ratio at 465 nm and 665 nm of the leaching solution; S502. Moisture content determination: Determined by the 105°C constant temperature drying method; S503. Nutrient content determination: The organic matter content is determined according to the NY / T304 - 1995 standard, the total nitrogen is determined by the Kjeldahl method, the total phosphorus is determined by the ammonium molybdate vanadate colorimetric method, and the total potassium is determined by the flame photometry method.

[0012] Preferably, the method optimizes the quality of pig manure compost through a specific microbial agent combination and is applicable to the treatment of agricultural waste and the preparation of organic fertilizers.

[0013] The present invention provides a method for improving the quality of pig manure compost using a specific microbial agent combination. It has the following beneficial effects: 1. The present invention significantly improves the passivation effect of heavy metals (such as Cu and Zn) during the composting process through a specific combination of microbial agents. This effect is due to the metabolites (such as organic acids and extracellular polysaccharides) produced by strains such as Bacillus licheniformis and Aspergillus niger. These substances can form stable complexes or precipitates with heavy metal ions, thereby reducing their bioavailability and decreasing the environmental risk when the compost product is applied to the soil.

[0014] 2. The present invention optimizes the retention and transformation of nutrients such as nitrogen, phosphorus, and potassium during the composting process through a specific combination of microbial agents. Because strains such as Bacillus subtilis and Bacillus megaterium decompose organic matter and fix nitrogen, reducing ammonia volatilization, while Aspergillus niger promotes the release of insoluble phosphorus, enhancing the availability of nutrients, providing technical support for the preparation of high-quality organic fertilizers.

[0015] 3. The present invention significantly improves the degree of compost maturity using a specific combination of microbial agents. The seed germination index, as an indicator of maturity, shows a 5.66% - 6.46% increase compared to the blank control group and a 7.8% increase compared to the commercial microbial agent group. The improvement is attributed to the synergistic effect of various microorganisms in the microbial agent. For example, Aspergillus niger decomposes complex organic matter, and Bacillus cereus accelerates cellulose degradation, accelerating the transformation process of organic matter into humus, making the compost product more suitable as an agricultural soil conditioner, while reducing the risk of phytotoxicity caused by unripe compost.

[0016] 4. The present invention controls the pig manure composting cycle within 30 - 35 days through a specific combination of microbial agents and a high-aerobic composting process, which is shorter compared to traditional composting methods (usually 45 - 60 days). The high-temperature active strains (such as Bacillus subtilis and Bacillus megaterium) in the microbial agent still maintain high activity when the pile temperature rises to 50℃ - 60℃, accelerating the decomposition of organic matter and microbial reproduction. Combined with the heat accumulation and air permeability design of the conical pile body, the fermentation efficiency is further improved, providing a time-based technical advantage for large-scale composting treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the composting method for a method of improving the quality of pig manure compost using a specific combination of microbial agents according to the present invention; Figure 2 It is a schematic diagram of the temperature curve for a method of improving the quality of pig manure compost using a specific combination of microbial agents according to the present invention; Figure 3 It is a schematic diagram of the pH value curve for a method of improving the quality of pig manure compost using a specific combination of microbial agents according to the present invention; Figure 4 It is a schematic diagram of the EC value curve for a method of improving the quality of pig manure compost using a specific combination of microbial agents according to the present invention; Figure 5Schematic diagram of the E4 / E6 curve for a method of improving the quality of pig manure compost using a specific bacterial agent combination in the present invention; Figure 6 Schematic diagram of the moisture content curve for a method of improving the quality of pig manure compost using a specific bacterial agent combination in the present invention; Figure 7 Schematic diagram of the determination of the seed germination index for a method of improving the quality of pig manure compost using a specific bacterial agent combination in the present invention; Figure 8 Schematic diagram of the seed germination index curve for a method of improving the quality of pig manure compost using a specific bacterial agent combination in the present invention; Figure 9 Schematic diagram of the organic matter curve for a method of improving the quality of pig manure compost using a specific bacterial agent combination in the present invention; Figure 10 Schematic diagram of the total nitrogen content determination curve for a method of improving the quality of pig manure compost using a specific bacterial agent combination in the present invention; Figure 11 Schematic diagram of the total phosphorus content determination curve for a method of improving the quality of pig manure compost using a specific bacterial agent combination in the present invention; Figure 12 Schematic diagram of the total potassium content determination curve for a method of improving the quality of pig manure compost using a specific bacterial agent combination in the present invention; Figure 13 Schematic diagram of the total carbon content determination curve for a method of improving the quality of pig manure compost using a specific bacterial agent combination in the present invention; Figure 14 Schematic diagram of the copper content determination curve for a method of improving the quality of pig manure compost using a specific bacterial agent combination in the present invention; Figure 15 Schematic diagram of the arsenic content determination curve for a method of improving the quality of pig manure compost using a specific bacterial agent combination in the present invention; Figure 16 Schematic diagram of the zinc content determination curve for a method of improving the quality of pig manure compost using a specific bacterial agent combination in the present invention; Figure 17 Stacked statistical chart of the detection of ARGs for a method of improving the quality of pig manure compost using a specific bacterial agent combination in the present invention; Figure 18 Pie chart of the antibiotic resistance categories of ARGs in the samples for a method of improving the quality of pig manure compost using a specific bacterial agent combination in the present invention; Figure 19 Stacked statistical chart of the detection of ARGs for a method of improving the quality of pig manure compost using a specific bacterial agent combination in the present invention; Figure 20 Pie chart of the antibiotic resistance categories of ARGs in the samples for a method of improving the quality of pig manure compost using a specific bacterial agent combination in the present invention; Figure 21 This is a statistical chart of the ARGs resistance mechanism for a method of improving the quality of pig manure compost using a specific combination of bacterial agents in the present invention. Detailed implementation manners

[0018] Next, in combination with the drawings of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0019] Please refer to the attached Figure 1 - attached Figure 7 , an embodiment of the present invention provides a method for improving the quality of pig manure compost using a specific combination of bacterial agents, including the following steps: S1. Preparation of compost raw materials: Select fresh pig manure and rice husk straw as compost raw materials. Among them, fresh pig manure provides organic substances and nutrients, and rice husk straw is used to adjust the air permeability and carbon-nitrogen ratio of the compost; S2. Preparation of bacterial agents: Prepare a specific combination of bacterial agents. The specific combination of bacterial agents includes Bacillus licheniformis, Bacillus megaterium, Aspergillus niger, Bacillus subtilis, and Bacillus cereus; S3. Composting process: Mix fresh pig manure, rice husk straw and the specific combination of bacterial agents evenly, and use the high-temperature aerobic composting method for composting; S4. Monitoring and regulation: During the composting process, monitor the temperature of the compost pile daily and turn the pile regularly to maintain aerobic conditions.

[0020] Specifically, in step S1, the selection of fresh pig manure needs to ensure that its moisture content is between 60% - 70% to provide a suitable microbial fermentation environment; if the moisture is too high, it can be adjusted by natural drying or adding dry rice husks. The addition amount of rice husk straw is usually controlled at 20% - 30% of the dry weight of pig manure to optimize the carbon-nitrogen ratio to within the range of 25:1 - 30:1, and it can be specifically adjusted according to the initial nitrogen content of pig manure determined by an elemental analyzer. In step S2, the specific combination of bacterial agents can be prepared by solid-state fermentation or liquid fermentation methods. Among them, for solid-state fermentation, rice bran or wheat bran is used as the matrix, the culture temperature is controlled at 28°C - 35°C, and the culture time is 48 - 72 hours; for liquid fermentation, a medium containing glucose and peptone is used, the pH value is adjusted to 6.5 - 7.5, the aeration rate is 0.5 - 1.0 vvm, and the culture time is 24 - 36 hours. The viable count of the bacterial agent needs to reach 108 -10 9 CFU / g or CFU / mL to ensure its activity in compost. In step S3, a mechanical stirring device (such as a turning machine) is recommended for the mixing process, and the mixing time is not less than 15 minutes to ensure uniform distribution of the microbial agent; the initial moisture content of the compost pile in aerobic composting is controlled at 55%-65%. If the moisture is insufficient, an appropriate amount of sterile water can be sprayed for adjustment. During the composting process, the ambient temperature should be maintained at 15℃-35℃ to avoid inhibition of microbial activity by extreme low or high temperatures. In step S4, the turning frequency can be dynamically adjusted according to the temperature of the compost pile. When the temperature exceeds 60℃, turn the pile every 2-3 days; when it is below 40℃, it can be extended to once every 5-7 days. When turning the pile, it is necessary to ensure full exchange of materials inside and outside the pile to maintain oxygen supply.

[0021] Please refer to the attached Figure 1 , in the specific microbial agent combination, the strain ratios of Bacillus licheniformis, Bacillus megaterium, Aspergillus niger, Bacillus subtilis, and Bacillus cereus are 3:3:3:3:1 or 3:3:2:2:1.

[0022] Specifically, the preparation of the strain ratios needs to be carried out under sterile conditions. Each strain can be cultured separately and then mixed in proportion, or co-fermented in the same culture medium. Bacillus licheniformis can be cultured through LB medium (containing 10g tryptone, 5g yeast extract, 10g sodium chloride per liter, pH 7.0), Bacillus megaterium and Bacillus subtilis use nutrient broth medium (containing 3g beef extract, 10g tryptone, 5g sodium chloride per liter, pH 7.2), Aspergillus niger uses PDA medium (containing 200g potatoes, 20g glucose, 15g agar per liter, pH 5.6), and Bacillus cereus is cultured in a starch-containing medium (such as 5g starch, 10g peptone, 5g sodium chloride per liter, pH 7.0). After each strain is cultured to the logarithmic growth phase, they are mixed in the ratio of 3:3:3:3:1 or 3:3:2:2:1. After mixing, a protective agent (such as 5%-10% glycerol or skimmed milk powder) can be added to maintain the activity of the strains. The ratio selection can be adjusted according to the heavy metal content of the compost raw materials. If the heavy metal (such as Cu, Zn) content is high, it is recommended to use the ratio of 3:3:2:2:1 because Aspergillus niger and Bacillus subtilis have stronger heavy metal passivation ability. The recommended dosage of the microbial agent is 0.05%-0.1% of the dry weight of the compost, that is, 0.5-1kg of the microbial agent is added per ton of dry weight material, and the optimal dosage can be determined through small-scale tests.

[0023] Please refer to the attached Figure 1 , in step S1, the fresh pig manure comes from a pig farm, and the rice husk straw is crushed to a particle size of 1-5 cm.

[0024] Specifically, the source of fresh pig manure should be healthy pigs to avoid manure containing antibiotic residues or pathogenic bacteria. After collection, it should be used within 24 hours to prevent anaerobic fermentation from producing odors or nutrient loss. The pretreatment of pig manure includes removing large impurities (such as stones and plastics) and preliminarily crushing it to a particle diameter of less than 5 cm to facilitate mixing with rice husk straw. The rice husk straw can be crushed using a hammer mill or a shredder, with the particle size controlled within 1 - 5 cm to ensure that it can increase the porosity of the compost pile and not cause the pile to be too dense due to being too fine. The crushed rice husk straw needs to be dried until the moisture content is below 15% to avoid excessive water absorption at the initial stage of composting, which may affect air permeability. If the source of rice husk straw is limited, it can also be partially replaced with corn straw or wheat straw, but attention should be paid to the difference in their carbon-nitrogen ratios (about 50:1 for corn straw and about 80:1 for wheat straw), and the addition ratio should be adjusted accordingly. When mixing, it is recommended to lay in layers, that is, first lay a layer of pig manure (about 20 - 30 cm thick), sprinkle a layer of rice husk straw (about 5 - 10 cm thick), and repeat the operation until the compost pile is formed to improve uniformity.

[0025] Please refer to the appendix Figure 1 , in step S3, in the high-temperature aerobic composting method, the height of the compost pile is controlled at 1.5 - 2 m, and the bottom diameter is controlled at 2 - 3 m to promote heat accumulation and air circulation.

[0026] Specifically, the settings of the height and bottom diameter of the compost pile need to be adjusted according to the site conditions and equipment capabilities. For small-scale composting (such as experimental piles), the height can be set at 1.5 m and the bottom diameter at 2 m, while for large-scale production piles, it can be extended to a height of 2 m and a bottom diameter of 3 m. When constructing the compost pile, it is recommended to lay a 10 - 15 cm thick layer of dry rice husk or wood chips at the bottom as a ventilation layer to avoid water accumulation at the bottom, which may affect aerobic fermentation. The top of the compost pile can be covered with materials with good air permeability (such as gunny sacks or straw curtains) to reduce water evaporation and the entry of external impurities, while maintaining heat accumulation. During the high-temperature aerobic composting process, the internal temperature of the compost pile usually rises to 50℃ - 60℃ on the 3rd - 5th day. This stage is the high-temperature period, and it is necessary to closely monitor to prevent the temperature from exceeding 65℃. Otherwise, the temperature can be reduced by increasing the turning frequency or spraying a small amount of water. The guarantee of air circulation can also be achieved by setting ventilation channels around the compost pile or using forced ventilation equipment (such as a blower, with an air volume of 0.1 - 0.3 m³ / min / ton), depending on the scale of the compost pile.

[0027] Please refer to the appendix Figure 1 , in step S3, the composting cycle is 30 - 35 days, and the compost pile is set in a conical shape.

[0028] Specifically, when constructing the conical pile, it is necessary to ensure uniform distribution of the bottom materials, which can be gradually piled up manually or mechanically (such as a loader). The top of the pile should be slightly pointed to facilitate the sliding of rainwater and avoid water accumulation at the top of the pile. The slope of the pile is recommended to be controlled between 45°-60°, which can not only ensure stability but also facilitate the aggregation of heat towards the center. The composting cycle of 30-35 days is applicable to summer or warm seasons (ambient temperature 20℃-30℃). If it is implemented in winter (ambient temperature below 15℃), it can be extended to 40-45 days, or a heat preservation material (such as a plastic film) can be covered outside the pile to maintain the activity of microorganisms. During the composting process, the first 10-15 days are the main fermentation stage. At this time, the organic matter decomposes rapidly, and the pile needs to be turned over 2-3 times a week; the last 15-20 days are the ripening stage, and the turning frequency can be reduced to once a week. After the cycle ends, the degree of ripeness can be preliminarily judged by visually observing the color of the compost (dark brown), smell (odorless), and texture (loose).

[0029] Please refer to the appendix Figure 1 - appendix Figure 16 , in step S4, the monitoring and regulation include: S401. Measure the temperatures at five positions (east, south, west, north, and center) of the pile using an alcohol thermometer every day, with the insertion depth of 20-30 cm, and record the ambient temperature; S402. On the 1st, 6th, 11th, 16th, 20th, 23rd, 29th, and 34th days of composting, take 30g samples from each of the five positions of the pile, mix them, and store them under the condition of -20℃.

[0030] Specifically, in step S401, the specific operation of temperature measurement should be carried out at 9:00-10:00 am every day to avoid the interference of daily temperature differences on the data. After the alcohol thermometer is inserted into the pile, it needs to stay for 1-2 minutes to stabilize the reading. If the temperature distribution of the pile is uneven (such as the center is more than 10℃ higher than the edge), it can be adjusted by local turning of the pile. The ambient temperature should be recorded using a hanging thermometer, placed 1 meter above the surrounding of the pile, and avoid direct sunlight affecting the accuracy. In step S402, sterile sampling spoons or gloves should be used for sample collection to avoid external microbial contamination. After each sampling, the tools should be cleaned with 75% alcohol. When mixing the samples, it is recommended to carry out the operation on a sterile workbench, gently grind them evenly with a sterile mortar, and squeeze out the air before putting them into a self-sealing bag to reduce oxidation. The samples stored in a -20℃ refrigerator should be tested within 30 days. If long-term storage is required, they can be transferred to a -80℃ ultra-low temperature refrigerator. The sampling points should cover different depths of the pile (such as the surface layer of 10 cm, the middle layer of 50 cm, and the bottom layer of 100 cm) to comprehensively reflect the composting process.

[0031] Please refer to the appendix Figure 1 - appendix Figure 6 , and also includes step S5, determination of physical and chemical indexes, specifically: S501. Determination of pH value, conductivity, and E4 / E6 value: Take 10.0 g of the compost sample and mix it with 100 mL of distilled water at a mass ratio of 1:10. After shaking at 160 r / min for 1 hour, filter it, and measure the pH value, conductivity, and the absorbance ratio at 465 nm and 665 nm of the leachate. S502. Moisture content determination: Use the constant temperature drying method at 105 °C for determination. S503. Nutrient content determination: The organic matter content is determined according to the standard NY / T304 - 1995, the total nitrogen is determined by the Kjeldahl method, the total phosphorus is determined by the ammonium vanadomolybdate colorimetric method, and the total potassium is determined by the flame photometry method.

[0032] Specifically, in step S501, when mixing the compost sample and distilled water, a 250 mL conical flask is required. Before shaking, the bottle mouth needs to be sealed to prevent volatilization. The shaking equipment can choose a constant temperature oscillator, and the temperature is set at 25 °C ± 1 °C. When filtering, first use double - layer gauze to remove large - particle residues, and then use a 0.45 μm filter membrane for fine filtration to ensure the clarity of the leachate. The pH value is measured using a precision pH meter (accuracy ±0.01), and it needs to be calibrated with standard buffer solutions (pH 4.00, 7.00, 10.00) before measurement; the conductivity is measured using a bench - type conductivity meter, and the probe needs to be rinsed 3 times with distilled water after cleaning; the E4 / E6 value is measured using an ultraviolet - visible spectrophotometer, and after wavelength calibration, it is measured three times and the average value is taken. In step S502, in the constant temperature drying method, the sample needs to be placed in an oven at 105 °C, and the drying time is generally 12 - 24 hours. Weigh it every 4 hours until the difference between two weighings is less than 0.01 g, which is regarded as constant weight. When cooling, it needs to be placed in a desiccator to avoid moisture absorption. In step S503, for organic matter determination, an oil bath heating device (temperature 165 °C ± 5 °C) is required, and the digestion time is 30 minutes; for total nitrogen determination, the digestion process needs to be carried out in a fume hood, add 10 mL of concentrated sulfuric acid and 0.5 g of catalyst, and heat until the solution is clear; for total phosphorus determination, a standard curve needs to be prepared, and the phosphorus concentration range is 0 - 10 mg / L; for total potassium determination, the sample needs to be diluted 50 times with deionized water to avoid clogging the flame photometer.

[0033] Please refer to the appendix Figure 1 The method optimizes the quality of pig manure compost through a specific combination of bacterial agents and is applicable to the treatment of agricultural waste and the preparation of organic fertilizers.

[0034] Specifically, the implementation site of this method should preferably be a flat and open area with good drainage, at least 50 meters away from water sources and residential areas to avoid odor or leachate pollution. After composting is completed, the product can be used as basal fertilizer for farmland or for vegetable planting. The recommended application rate per mu is 1 - 2 tons. When used in combination with chemical fertilizers, the amount of chemical fertilizers can be reduced by 20% - 30%. The optimized compost product can also be processed into granular fertilizer or liquid fertilizer. For the production of granular fertilizer, a granulator (rotation speed 30 - 50 r / min) is required, and the moisture content should be controlled below 10% after drying; for liquid fertilizer, the active ingredients are extracted by leaching and then diluted and sprayed on the crop leaves. To improve economic benefits, the microbial agent formula can be adjusted according to the local soil nutrient requirements, such as increasing the proportion of Bacillus subtilis to improve nitrogen fixation ability, or increasing the proportion of Aspergillus niger to enhance phosphorus release effect. During the implementation of the method, it is recommended to record the impact of daily weather conditions (such as rainfall, humidity) on the composting process to optimize subsequent operations.

[0035] The effects of improving the compost quality of the above - mentioned method include: The seed germination index is increased by 5.66% - 6.46% compared with the blank control group and by 7.8% compared with the commercial microbial agent group; The total nitrogen loss is reduced by 43.74% - 58.66% compared with the blank control group and by 26.28% - 41.2% compared with the commercial microbial agent group; The total phosphorus content is increased by 7.06% - 20.86% compared with the blank control group and by 16.34% - 30.14% compared with the commercial microbial agent group; The total potassium loss is reduced by 3.7% - 3.71% compared with the blank control group.

[0036] The passivation ability of specific microbial agent combinations for heavy metals includes: The passivation rate for heavy metal Cu is increased by 27.35% - 38.56% compared with the blank control group and by 51.61% - 62.82% compared with the commercial microbial agent group; The passivation rate for heavy metal Zn is increased by 88.13% - 99.14% compared with the blank control group and by 54.6% - 65.61% compared with the commercial microbial agent group.

[0037] Please refer to Appendix Figure 7 - Appendix Figure 21 , and four samples are taken from each of the four periods of pig manure composting for quantitative analysis of antibiotic resistance genes. The qpcr high - throughput technology is used for the quantitative analysis of antibiotic resistance genes. There are a total of four groups, and four samples are taken from each group. The sample numbers are CK1, CK3, CK5, CK7, COM1, COM3, COM5, COM7, 0.5A1, 0.5A3, 0.5A5, 0.5A7, and 0.5B1, 0.5B3, 0.5B5, 0.5B7 respectively.

[0038] Such asFigure 18 A total of 296 drug resistance genes were detected, and 231 drug resistance genes were detected. There were 14 mobile genetic elements, including transposase and integron, accounting for 5.07% and 0.72% respectively. The proportions of multidrug resistance (Multidrug) were 23.91%; macrolide-lincosamide-streptogramin B (MLSB) were 15.22%; aminoglycoside and tetracycline were both 13.04%; beta-lactamase (Beta_Lactamase) were 12.32%; vancomycin (Vancomycin) were 9.42%; others were 2.92%; chloramphenicol and sulfonamide were both 2.17%.

[0039] As the core vector for the horizontal transfer of drug resistance genes, mobile genetic elements showed significant activity in this detection: the high detection rate of transposase genes (5.07%) revealed the active transposition behavior of DNA transposons, and such elements could promote the jumping of drug resistance genes between chromosomes and plasmids through the "cut-paste" mechanism; although integrons (0.72%) had the lowest proportion, as the key vector of the gene cassette capture system, they could efficiently integrate drug resistance genes such as beta-lactamase and aminoglycoside, accelerating the formation of multi-drug resistance phenotypes. The two were directly associated with the high proportion of multidrug resistance genes (23.91%), jointly constituting the molecular basis of drug resistance transmission.

[0040] Figure 19 The detected amounts and compositional distributions of antibiotic resistance genes (ARGs) in different treatment groups (0.5a group, 0.5b group, blank control group, commercial microbial agent group) were shown.

[0041] Note: Sample: sample name; Amino: Aminoglycoside; Beta: Beta_Lactamase; Chlor: Chloramphenicol; Sul: Sulfonamide; Tet: Tetracycline; Van: Vancomycin. The table header mainly consists of two parts, Sample: sample name; the rest are the antibiotic categories against which the antibiotic resistance genes resist. The meaning of each number: the number of ARGs detected in sample Sample that belong to the resistance against a certain antibiotic.

[0042] From the overall trend:[[]]END]] In this experiment, the total amount of detected drug-resistant genes in the 0.5a group and the 0.5b group in the first sample on the first day of the experiment was significantly higher than that in the commercial microbial agent group (com group) and the blank control group (ck group). From the perspective of dynamic changes, the detected amount of drug-resistant genes in the 0.5a group and the 0.5b group showed an overall downward trend over time. Among them, the detected amount of drug-resistant genes in the 0.5a group decreased from 168 at A1 to 139 at A7, with a total reduction of 29; the 0.5b group decreased from 171 at B1 to 136 at B7, with a total reduction of 35, indicating that the content of drug-resistant genes in these two groups gradually decreased over time. On the contrary, the detected amount of drug-resistant genes in the blank control group (ck group) showed an increasing trend, increasing from 71 at CK1 to 142 at CK7, with a total increase of 71, and the detected amount of drug-resistant genes continued to rise. The detected level of drug-resistant genes in the commercial microbial agent group (com group) remained relatively stable from the start to the end of the experiment, ranging from 144 at C1 to 147 at C7, with only a 3-point fluctuation throughout the process, and there was no obvious change in the detected level of drug-resistant genes during the experiment.

[0043] From the perspective of the detection of individual drug-resistant genes: Aminoglycoside: In the 0.5a group (from A1 to A7), it decreased from 27 to 18, and in the 0.5b group (from B1 to B7), it decreased from 27 to 18, both showing a decreasing trend; in the blank control group (ck group), it increased significantly from 11 at CK1 to 24 at CK7, showing an increasing trend; in the commercial microbial agent group (com group), it fluctuated slightly between C1 (25) and C7 (20), remaining relatively stable overall. Multidrug: In the 0.5b group, it decreased from 34 at B1 to 31 at B7, showing a slight decrease; in the ck group, it increased significantly from 11 at CK1 to 28 at CK7, showing an obvious increasing trend; in the com group, it increased slightly from 25 at C1 to 29 at C7, with a small change range. Beta_Lactamase: In the 0.5a group, it decreased from 22 at A1 to 17 at A7, and in the 0.5b group, it decreased from 24 at B1 to 20 at B7; in the ck group, it increased from 7 at CK1 to 24 at CK7, with a significant increase; in the com group, it remained stable between C1 (23) and C7 (25). Tetracycline: In the 0.5a group, it decreased from 20 at A1 to 18 at A7, and in the 0.5b group, it decreased from 22 at B1 to 18 at B7; in the ck group, it increased from 9 at CK1 to 13 at CK7; in the com group, it increased from 13 at C1 to 19 at C7, showing a slow increase. For other genes such as Integron, the detected amounts in each group were generally low (mostly 1–3). Although there were slight fluctuations over time in the ck group (such as from 2 at CK1 to 2 at CK7), the overall change trend was not significant.

[0044] The results showed that the 0.5a group and the 0.5b group showed significant inhibitory effects on six types of drug-resistant genes. Among them, the decreases in aminoglycoside, beta-lactamase, and macrolide-lincosamide-streptogramin B (MLSB) were the most prominent (for example, aminoglycoside decreased from 27 to 18 in the 0.5a group and from 27 to 18 in the 0.5b group). In addition, tetracycline, transposase, and vancomycin also showed varying degrees of decreasing trends. Compared with the significant increase in the detection amount of drug-resistant genes in the blank control group (ck group) (for example, aminoglycoside increased from 11 to 24), the inhibitory effect of the 0.5a / 0.5b group may be related to the improvement of microbial metabolic activity, the regulation of drug-resistant gene expression, or the reduction of horizontal transfer. The regulatory effect of the commercial microbial agent group (com group) on most drug-resistant genes was relatively stable.

[0045] Detection results of ARG resistance mechanisms From the perspective of the proportional distribution of drug-resistant gene resistance mechanisms, as Figure 20 shown, efflux pump (efflux pump type) accounted for the highest proportion at 34.78%, indicating that the drug-resistant mechanism of bacteria actively expelling antibiotics through efflux pumps was the most prominent in this research object. Secondly, antibiotic deactivate (antibiotic inactivation type, 33.33%) had a significant proportion, reflecting that the strategy of bacteria achieving drug resistance by enzymatically degrading antibiotics was also common; cellular protection (cellular protection type, 24.64%) had a relatively high proportion, indicating that the phenomenon of bacteria resisting antibiotics by enhancing cellular defense ability was also relatively common. Relatively speaking, integrase (integrase type, 0.72%) and other / unknown (other / unknown type, 1.45%) had extremely low proportions, indicating that these mechanisms had limited effects on the current analysis object; while transposase (transposase type, 5.07%) had a moderate proportion, suggesting that the transposase-mediated transfer of drug-resistant genes had a certain role. The overall distribution reflected the diversity of drug-resistant mechanisms. The high proportions of efflux pump type and antibiotic inactivation type highlighted the complexity of bacterial drug-resistant mechanisms and the challenges of prevention and control work.

[0046] Figure 20 showed the distribution and changes in the detection amount of drug-resistant gene (ARG) resistance mechanisms in different treatment groups (0.5a group, 0.5b group, blank control group, commercial microbial agent group).

[0047] Overall trend: Total amount comparison on the first day: On the first day of the experiment, the total detection amount of the drug resistance gene resistance mechanism in the 0.5a group (total amount of A1 is 164) and the 0.5b group (total amount of B1 is 166) was significantly higher than that in the commercial microbial agent group (total amount of C1 is 148) and the blank control group (total amount of CK1 is 74). The 0.5a group and the 0.5b group showed a downward trend: the 0.5a group decreased from 164 of A1 to 138 of A7, with a total reduction of 26; the 0.5b group decreased from 166 of B1 to 141 of B7, with a total reduction of 25, indicating that the detection amount of the drug resistance gene resistance mechanism in the two groups gradually decreased over time. The blank control group (ck group) showed an increasing trend, increasing from 74 of CK1 to 142 of CK7, with a total increase of 68, and the detection amount of the resistance mechanism continued to rise. The commercial microbial agent group (com group) remained stable, from 148 of C1 to 144 of C7, with only a fluctuation of 4 throughout the process and no significant change during the experiment.

[0048] Analysis of individual drug resistance gene resistance mechanisms: antibiotic deactivate (antibiotic inactivation type): the 0.5a group decreased from 61 of A1 to 46 of A7; the 0.5b group decreased from 66 of B1 to 51 of B7; the ck group increased from 26 of CK1 to 57 of CK7; the com group fluctuated slightly between C1 (57) and C7 (56). efflux pump (efflux pump type): the 0.5a group decreased from 51 of A1 to 47 of A7; the 0.5b group decreased from 50 of B1 to 48 of B7; the ck group increased from 18 of CK1 to 42 of CK7; the com group increased from 39 of C1 to 45 of C7, showing a slow increase. cellular protection (cell protection type): the 0.5a group decreased from 40 of A1 to 34 of A7; the 0.5b group decreased from 35 of B1 to 32 of B7; the ck group increased from 21 of CK1 to 30 of CK7; the com group decreased from 38 of C1 to 33 of C7. For others such as integrase (integrase type), the detection amount in each group was generally low (mostly 1 - 2), and the change trend was not significant.

[0049] The 0.5a group and the 0.5b group showed inhibitory effects on the resistance mechanisms of antibiotic deactivation, efflux pump, cellular protection and other drug-resistant genes. Among them, the decline in the antibiotic inactivation type and the efflux pump type was more prominent (for example, antibiotic deactivation decreased from 61 to 46 in the 0.5a group and from 66 to 51 in the 0.5b group). Compared with the significant increase in the detection amount of the resistance mechanism in the blank control group (ck group) (for example, antibiotic deactivation increased from 26 to 57), the inhibitory effect of the 0.5a / 0.5b group may be related to the regulation of the microbial community and the inhibition of the expression of drug-resistant related genes under the treatment conditions; the regulation effect of the commercial microbial agent group (com group) on the resistance mechanisms of most drug-resistant genes was relatively stable and no significant fluctuations occurred.

[0050] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for improving the quality of pig manure composting by using a specific bacterial agent combination, characterized in that: The following steps are involved: S1. Preparation of compost raw materials: fresh pig manure and rice husk straw are selected as compost raw materials, wherein the fresh pig manure provides organic matter and nutrients, and the rice husk straw is used to adjust the air permeability and carbon-nitrogen ratio of the compost; S2. preparing a bacterial agent, preparing a specific bacterial agent combination, wherein the specific bacterial agent combination comprises Bacillus licheniformis, Bacillus megaterium, Aspergillus niger, Bacillus subtilis and Bacillus cereus; S3, composting process, mixing fresh pig manure, rice husk straw and the specific bacterial agent combination evenly, and composting by high temperature aerobic composting method; S4. Monitoring and control: During the composting process, the temperature of the pile should be monitored daily and the pile should be turned regularly to maintain aerobic conditions.

2. The method for improving the quality of pig manure composting by using a specific bacterial agent combination according to claim 1, characterized in that: The strain ratio of Bacillus licheniformis, Bacillus megaterium, Aspergillus niger, Bacillus subtilis and Bacillus cereus in the specific bacterial agent combination is 3:3:3:3:1 or 3:3:2:2:

1.

3. The method for improving the quality of pig manure composting by using a specific bacterial agent combination according to claim 1, characterized in that: In step S1, the fresh pig manure comes from a pig farm, and the rice husk straw is crushed to a particle size of 1-5 cm.

4. The method for improving the quality of pig manure composting by using a specific bacterial agent combination according to claim 1, characterized in that: In step S3, the height of the pile in the high-temperature aerobic composting method is controlled at 1.5-2 meters, and the bottom diameter is controlled at 2-3 meters to promote heat accumulation and air circulation.

5. The method for improving the quality of pig manure composting by using a specific bacterial agent combination according to claim 1, characterized in that: In step S3, the composting cycle is 30-35 days, and the compost body is set in a cone shape.

6. The method for improving the quality of pig manure composting by using a specific bacterial agent combination according to claim 1, characterized in that: In step S4, the monitoring and regulation includes: S401. Use an alcohol thermometer to measure the temperature of the pile in the east, south, west, north and center every day, insert it to a depth of 20-30 cm, and record the ambient temperature; S402. On the 1st, 6th, 11th, 16th, 20th, 23rd, 29th and 34th days of composting, take 30g samples from five directions of the compost pile, mix them and store them at -20℃.

7. The method for improving the quality of pig manure composting by using a specific bacterial agent combination according to claim 1, characterized in that: The step S5 is also included, which is to measure the physical and chemical indicators, specifically: S501, pH value, conductivity and E4 / E6 value determination: 10.0 g of compost sample was mixed with 100 mL of distilled water at a mass ratio of 1:10, shaken at 160 r / min for 1 hour, filtered, and the pH value, conductivity and absorbance ratio at 465 nm to 665 nm of the extract were determined; S502, moisture content determination, using 105℃ constant temperature drying method; S503, nutrient content determination, organic matter content is determined according to NY / T304-1995 standard, total nitrogen is determined by Kjeldahl method, total phosphorus is determined by ammonium vanadate molybdate colorimetry, and total potassium is determined by flame photometry.

8. The method for improving the quality of pig manure composting by using a specific bacterial agent combination according to claim 1, characterized in that: The method optimizes the quality of pig manure composting through a specific bacterial agent combination and is suitable for agricultural waste treatment and organic fertilizer preparation.

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