A method for preparing organic fertilizer through grapevine branch-cow dung-microbial agent synergistic fermentation

Abstract

The application discloses a method for preparing organic fertilizer through grapevine branch-cow dung-microbial agent synergic fermentation, and belongs to the technical field of agricultural waste resource utilization and organic fertilizer preparation. The method comprises the following steps: after the grapevine branches are crushed, the grapevine branches are mixed with the cow dung at a mass ratio of 1: (1-2), microbial agents are added for composting, and organic fertilizer is obtained; and potting experiments are conducted to verify the fertilizer efficiency of the organic fertilizer. The method for preparing organic fertilizer through grapevine branch-cow dung-microbial agent synergic fermentation can convert grapevine branches into high-quality organic fertilizer, solves the problems of high lignification degree of grapevine branches, slow degradation of the grapevine branches alone, and long composting and decomposition period of the grapevine branches, improves the defects of imbalance of carbon and nitrogen ratio and high nitrogen loss rate of the cow dung alone, and improves the adaptability of composting raw materials by optimizing the mixing ratio of the grapevine branches and the cow dung. The method provides a typical example for agricultural waste resource utilization and promotes the development of circular agriculture.

Description

Technical Field

[0001] This invention relates to the field of agricultural waste resource utilization and organic fertilizer preparation technology, and in particular to a method for preparing organic fertilizer through synergistic fermentation of grape branches, cow dung, and microbial agents. Background Technology

[0002] In the process of grape cultivation and production management, grapevines need to be pruned and shaped every year, generating nearly ten million tons of grape branch waste. Currently, these grape branches are mostly disposed of by open burning or indiscriminate dumping and rotting, which not only causes serious environmental pollution and safety hazards, but also leads to a huge waste of resources. According to relevant research, directly burning 1 ton of grape branches in the open will release 51 kg of CO2 and 5.1 kg of total suspended particulate matter into the atmosphere. At the same time, indiscriminately discarded grape branches will pollute the soil, occupy land resources, and disrupt the ecological balance, creating a dual challenge of environmental protection and the resource utilization of organic materials.

[0003] Grapevine branches are rich in organic matter such as cellulose and hemicellulose, making them a high-quality raw material for organic fertilizer. They are also widely available and inexpensive. Applying organic fertilizer made from grapevine branches can effectively increase soil porosity, improve soil structure, and enhance soil water and fertilizer retention capacity. The beneficial microorganisms they contain can also promote soil nutrient transformation and absorption, inhibit pathogen growth, and reduce soil-borne diseases. This is of great significance for building a green and healthy soil ecosystem and promoting sustainable agricultural development. Furthermore, large-scale preparation and application can significantly reduce agricultural production costs. However, grapevine branches have a high lignin content (approximately 15-20%), a high degree of lignification, and a slow degradation rate (a decomposition period of over 60 days). This results in extremely low microbial degradation efficiency and a long decomposition period, severely impacting production efficiency and hindering large-scale resource utilization.

[0004] Cow manure is rich in nutrients such as nitrogen, phosphorus, and potassium, and is one of the traditional raw materials for organic fertilizer. However, the carbon-to-nitrogen ratio of cow manure is relatively low (about 10~17:1), and ammonia volatilization is likely to occur during composting. The nitrogen loss rate can reach more than 30%, and the organic matter content in the finished organic fertilizer is low, which makes it difficult to meet the needs of efficient crop fertilization and limits its resource utilization effect.

[0005] Therefore, developing a process that can efficiently decompose grape branches and cow manure, improve the quality of organic fertilizer, and achieve efficient resource utilization of both has become an urgent technical problem to be solved. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing organic fertilizer by co-fermentation of grape branches and cow manure, so as to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is an organic fertilizer, the raw materials of which include grape branches, cow manure and microbial agents; The mass ratio of grapevines to cow dung is 1:(1~2).

[0008] Controlling the mass ratio of grape vines to cow manure to 1:(1~2) can meet the needs of microbial degradation.

[0009] Preferably, the amount of microbial inoculant added is 0.50~0.54% of the total mass of grape vines and cow manure.

[0010] Preferably, the microbial agent includes MC composting agent or T composting agent; The amount of MC composting agent added is 0.50% of the total mass of grapevines and cow manure; The amount of the T composting agent added is 0.54% of the total mass of grapevines and cow manure.

[0011] Preferably, the main components of the MC composting agent include Bacillus licheniformis, Pseudomonas beichengensis, and Bacillus pantothenica, etc. The main components of the T-composting agent include brewer's yeast, Trichoderma viride, and Bacillus subtilis.

[0012] The second technical solution of the present invention: a method for preparing organic fertilizer through synergistic fermentation of grape branches, cow dung, and microbial inoculants, comprising the following steps: Grape branches are crushed and mixed with cow manure at a mass ratio of 1:(1~2), and microbial agents are added for composting to obtain the organic fertilizer.

[0013] Preferably, the length of the crushed grape branches is 0.01~1cm.

[0014] Preferably, the parameters of the compost include: turning frequency of 3 to 5 days / time and moisture content of the compost pile of 50 to 65%.

[0015] The present invention discloses the following technical effects: (1) The organic fertilizer containing grape branches and cow manure of the present invention is rich in nutrients and can meet the growth needs of crops.

[0016] (2) The method of preparing organic fertilizer by co-fermentation of grape branches, cow manure, and microbial agents of the present invention can transform grape branches into high-quality organic fertilizer, solving the problems of high lignification degree of grape branches, slow degradation when composted alone, and long composting cycle. It also improves the defects of unbalanced carbon-nitrogen ratio and high nitrogen loss rate in cow manure composting alone. By optimizing the mixing ratio of grape branches and cow manure, the composting raw material composting adaptability is improved. By further introducing special composting agents (i.e., microbial agents), the problems of local anaerobic and unstable temperature in mixed composting are solved, effectively solving the problem of waste treatment. The method of the present invention can not only reduce environmental pollution, but also realize the circular economy model of "resource-product-recycled resources", which is in line with the national "dual carbon" strategic goal, provides a typical example for the resource utilization of agricultural waste, and promotes the development of circular agriculture.

[0017] (3) The method of the present invention can effectively utilize discarded grape branches, avoid the pollution and waste of grape branches to the environment, and provide a new idea for green and sustainable agricultural development.

[0018] (4) This invention breaks through the limitations of traditional single treatment or simple mixed composting of agricultural waste, and innovatively constructs a closed-loop resource recycling path of "grape branches - cow manure - organic fertilizer - farmland", thereby realizing the efficient resource utilization of two types of agricultural waste, grape branches and cow manure. Its core innovations are: First, it proposes a precise ratio scheme of grape branches and cow manure, and promotes the synergistic degradation of the two raw materials in the composting process by scientifically matching their carbon-nitrogen ratio and material characteristics; Second, it selects and adds special composting agents (i.e. microbial agents) that are beneficial to the lignification characteristics of grape branches, which can efficiently break down the lignified structure and accelerate the decomposition of organic matter; Third, it establishes an integrated composting process control system that includes parameters such as grape branch crushing particle size, composting fermentation temperature, and turning frequency, providing a stable and suitable environment for the composting process; Fourth, it analyzes the nutrient retention through physicochemical test results to ensure that the finished organic fertilizer is both thoroughly composted and retains effective nutrients to the greatest extent. Attached Figure Description

[0019] 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.

[0020] Figure 1 The temperature changes during composting in Example 1 and the effect of organic fertilizer on seed germination rate at different times are shown in (a) and (b) respectively. Figure 2The table shows the changes in pH and conductivity during different composting periods in Example 1, where (a) represents pH and (b) represents conductivity. Figure 3 The changes in available nutrient content during composting in Example 1 are shown, where (a) is available nitrogen, (b) is available phosphorus, and (c) is available potassium. Figure 4 The changes in total nutrient content during composting in Example 1 are shown, where (a) represents total nitrogen, (b) represents total phosphorus, and (c) represents organic matter. Figure 5 This is a two-factor analysis diagram of the physicochemical indicators of the last sample taken from the compost plant in Example 1; Figure 6 The above are stacked bar charts showing the relative abundance of microbial community phyla at the last sampling level in Example 1. Among them, (a) is a stacked bar chart showing the relative abundance of bacterial community phyla at the level under different ratios of grape vines and cow manure, and (b) is a stacked bar chart showing the relative abundance of fungal community phyla at the level under different ratios of grape vines and cow manure. Figure 7 The images show the ASVs species and PCA diagrams of the microbial community of the last sampled microorganism in Example 1. Among them, (a) shows the common ASV species of soil bacteria under different ratios of grape vines and cow manure, (b) shows the common ASV species of soil fungi under different ratios of grape vines and cow manure, (c) shows the PCA diagram of the bacterial community under different ratios of grape vines and cow manure, and (d) shows the PCA diagram of the fungal community under different ratios of grape vines and cow manure. Figure 8 The above are the alpha abundance diagrams of the microbial community in Example 1, where (a) is the alpha abundance diagram of the bacterial community under different ratios of grape vines and cow manure, and (b) is the alpha abundance diagram of the fungal community under different ratios of grape vines and cow manure. Figure 9 This is a growth diagram of the potted plant in Example 1. Detailed Implementation

[0021] 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.

[0022] 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. Every smaller range between any stated value or intermediate value within a stated range, and 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0027] In the specific embodiments of this invention, the experimental site was the Agricultural Science Training Base of Ningxia University; the grape branches were taken from Meihe Winery on the eastern foothills of Helan Mountain in Ningxia Hui Autonomous Region (longitude 106°13'E, latitude 38°49'N); and the fresh cow manure was taken from a cattle farm in Helan County, Ningxia Hui Autonomous Region.

[0028] Information regarding the source of the microbial inoculants used in composting is as follows: MC composting agent (i.e., MC) is produced by Ningxia Dairy Cattle Research Institute (Co., Ltd.). The agent consists of 7 strains of bacteria, in the following order: Bacillus licheniformis (…). Bacillus licheniformis ), Xanthomonas cirrhosa ( Xanthomonas Cynarae ), Beicheng Pseudomonas ( Pseudomonas boreopoles ), pantothenic acid mycobacterium ( Virgibacillus pantothenticus ), Galactobacter ( Bacillus galactophilus ), Bordetella ( Bordetella bronchiseptica ) and Pseudomonas ( Pseudoxanthomonas taiwanensis Beihai Qiangxing Biotechnology Co., Ltd.'s T composting agent (i.e., T) mainly consists of beneficial microorganisms such as Saccharomyces cerevisiae, Bacillus subtilis, Bacillus licheniformis, and Trichoderma viride, with an effective live bacteria count ≥200 million / g.

[0029] Example 1 A method for preparing organic fertilizer through co-fermentation of grape vines and cow manure: (1) In the vineyard of Miho Winery, the dried winter pruning branches of grapes are collected, and impurities such as gravel mixed in with the branches are removed. The branches are then crushed into 0.01~1cm (excluding 0) size using a crusher to obtain grape branch fragments.

[0030] (2) N1 treatment: The experiment started on May 15, 2025. Grape branch fragments and fresh cow manure were mixed at a mass ratio of 1:1. MC composting agent was added (the amount of MC composting agent added was 0.50% of the total mass of grape branches and cow manure). After mixing evenly, the mixture was placed in a composting device (PVC plastic bucket, volume of 25L) for aerobic composting. During the composting period, a rainproof cloth was used to cover the compost to prevent rainwater and other external environmental influences. The compost was turned over and mixed every 3 to 5 days to ensure sufficient oxygen supply. Water was replenished regularly to keep the moisture content between 50% and 65% during the composting period.

[0031] N2 treatment: The experiment began on May 15, 2025. Grape twig fragments and fresh cow manure were mixed at a 1:1 mass ratio, and T composting agent was added (the amount of T composting agent added was 0.54% of the total mass of grape twigs and cow manure). After being mixed evenly, the mixture was placed in a composting device (PVC plastic bucket, volume 25L) for aerobic composting. During composting, a rainproof cloth was used to cover the compost to prevent rainwater and other external environmental influences. The compost was turned and mixed every 3 to 5 days to ensure sufficient oxygen supply. Moisture was added regularly to keep the moisture content between 50% and 65% during composting.

[0032] N3 treatment: The experiment began on May 15, 2025. Grape twig fragments and fresh cow manure were mixed evenly at a 1:1 mass ratio and placed in a composting device (PVC plastic bucket, volume 25L) for aerobic composting. During composting, a rainproof cloth was used to cover the container to prevent rain and other external environmental influences. The compost was turned and mixed every 3 to 5 days to ensure sufficient oxygen supply. Water was added regularly to keep the moisture content between 50% and 65% during composting.

[0033] N4 treatment: The experiment began on May 15, 2025. Grape twig fragments and fresh cow manure were mixed at a mass ratio of 1:2. MC composting agent was added (the amount of MC composting agent added was 0.50% of the total mass of grape twigs and cow manure). After being mixed evenly, the mixture was placed in a composting device (PVC plastic bucket, volume 25L) for aerobic composting. During composting, a rainproof cloth was used to cover the container to prevent rainwater and other external environmental influences. The compost was turned and mixed every 3-5 days to ensure sufficient oxygen supply. Moisture was added regularly to maintain the moisture content between 50% and 65% during the composting period.

[0034] N5 Treatment: The experiment began on May 15, 2025. Grape twig fragments were mixed with fresh cow manure at a mass ratio of 1:2. T composting agent was added (the amount of T composting agent added was 0.54% of the total mass of grape twigs and cow manure). After being mixed evenly, the mixture was placed in a composting device (PVC plastic bucket, volume 25L) for aerobic composting. During composting, the container was covered with a rainproof cloth to prevent rainwater and other external environmental influences. The container was turned and mixed every 3 to 5 days to ensure sufficient oxygen supply. Moisture was added regularly to keep the moisture content between 50% and 65% during composting.

[0035] N6 treatment: The experiment began on May 15, 2025. Grape twig fragments and fresh cow manure were mixed evenly at a mass ratio of 1:2 and then placed in a composting device (PVC plastic bucket, volume 25L) for aerobic composting. During the composting period, a rainproof cloth was used to cover the container to prevent rainwater and other external environmental influences. The compost was turned and mixed every 3 to 5 days to ensure sufficient oxygen supply. Water was added regularly to keep the moisture content between 50% and 65% during the composting period.

[0036] N7 Treatment: The experiment began on May 15, 2025. MC composting agent (0.50% of the total mass of grapevines and cow manure) was added to fresh cow manure and mixed thoroughly. The mixture was then placed in a composting device (PVC plastic bucket, 25L volume) for aerobic composting. During composting, a rainproof cloth was used to cover the compost to prevent rain and other external environmental influences. The compost was turned and mixed every 3-5 days to ensure sufficient oxygen supply. Moisture was added regularly to maintain the moisture content between 50-65% during composting.

[0037] N8 Treatment: The experiment began on May 15, 2025. T composting agent (0.54% of the total mass of grapevines and cow manure) was added to fresh cow manure. After being mixed evenly, the mixture was placed in a composting device (PVC plastic bucket, 25L volume) for aerobic composting. During composting, the compost was covered with a tarpaulin to prevent rain and other external environmental influences. The compost was turned and mixed every 3-5 days to ensure sufficient oxygen supply. Moisture was added regularly to keep the moisture content between 50-65% during composting.

[0038] N9 treatment: The experiment began on May 15, 2025. Fresh cow manure was placed in a composting device (PVC plastic bucket, volume 25L) for aerobic composting. During the composting period, the container was covered with a tarpaulin to prevent rain and other external environmental influences. The compost was turned and mixed every 3 to 5 days to ensure sufficient oxygen supply. Moisture was added regularly to keep the moisture content between 50% and 65% during the composting period.

[0039] During composting, unified management was implemented. Temperature was measured daily between 5 and 6 PM using a probe thermometer, with measurements taken at the lower and upper parts of the composting process. The average value was recorded as the daily composting temperature. Samples were taken weekly during composting. One portion was air-dried in the laboratory, while another portion was stored in a -20°C refrigerator (for later supplementary sample testing and index determination). In accordance with the organic fertilizer (NY T 525-2021) standard, changes in temperature, seed germination index (GI), pH, EC (electrical conductivity), total nitrogen (TN), total phosphorus, available nitrogen, available phosphorus, available potassium, and organic matter were measured during the composting process.

[0040] Table 1 Experimental Design for Organic Fertilizer Preparation The physicochemical properties of organic fertilizer were determined as follows: Collected samples were air-dried, ground, and sieved through 1mm and 0.25mm sieves. The contents of total nitrogen, total phosphorus, alkaline-available nitrogen, available phosphorus, available potassium, and organic matter were determined according to the organic fertilizer standard (NY T 525-2021). Fertilizer pH (water-to-fertilizer ratio 5:1) was measured using a PHS-25 precision acidity meter; EC value (water-to-soil ratio 5:1) was measured using a conductivity meter; organic matter was measured using a soil organic carbon analyzer; total nitrogen was determined using the Kjeldahl method; total phosphorus was determined using the molybdenum-antimony colorimetric method; alkaline-available nitrogen was determined using the alkaline diffusion method; available phosphorus was determined using the molybdenum-antimony colorimetric method; and available potassium was determined using flame photometry. During organic fertilizer preparation, samples were taken every 10 days for seed germination rate (GI) testing. The seed germination rate indicated whether the organic fertilizer was fully decomposed.

[0041] (I) Analysis of temperature changes and causes during composting The temperature changes during composting and the effects of organic fertilizer at different stages on seed (cucumber seed) germination rate are shown in [reference needed]. Figure 1 , where (a) is the composting temperature and (b) is the seed germination rate.

[0042] Figure 1 The room temperature mentioned refers to the natural temperature changes during composting.

[0043] from Figure 1As can be seen, there are significant differences in compost temperature and the duration of high temperature maintenance among different treatments. The grouping by substrate ratio is as follows: In the grapevine vine + cow manure 1:1 group, treatments N1, N2, and N3 entered the high-temperature stage (≥50℃) on days 12, 14, and 16, respectively, with high-temperature maintenance times of 10, 8, and 6 days, and maximum temperatures of 52.4℃, 53.4℃, and 52.4℃, respectively. Overall, N1>N2>N3; In the grapevine vine + cow manure 1:2 group, treatment N4 showed the longest temperature increase. The fastest (entering high temperature on day 7), the highest temperature was maintained for 15 days with a maximum temperature of 58.1℃, which was the best among all treatments. The N5 and N6 treatments entered high temperature on days 8 and 10, respectively, and maintained high temperature for 12 and 9 days, with maximum temperatures of 56.4℃ and 53.8℃, respectively. Within the same group, the treatment with added microbial agent was better than the treatment without microbial agent. The pure cow manure group (N7~N9) showed consistent performance, all entering high temperature around day 15 and maintaining it for 7 days, with a maximum temperature of about 51℃, which was significantly lower than the treatment with added grape branches.

[0044] The overall trend of compost temperature was influenced by the substrate ratio, exogenous microbial agents, and the composting process. The 1:2 ratio of grape twigs to cow manure was the optimal treatment in this experiment, exhibiting strong microbial activity, a long high-temperature period, and a high peak value. Treatments with added microbial agents (N1, N2, N4, N5, N7, N8) maintained high temperatures for significantly longer than the aseptic group. MC and T composting agents prolonged the high-temperature period by promoting organic matter decomposition and regulating microbial metabolism. Temperature rose rapidly in the initial stage of composting (0-15 days), gradually decreased in the middle stage (15-30 days), and stabilized in the later stage (30-40 days). Treatments N4, N5, and N2 showed the best performance, each achieving stable heat production through appropriate substrate ratios and the action of the microbial agents.

[0045] Samples from each treatment were taken once every 10 days to conduct a seed germination rate test. Water culture was set as a control (CK) to prove that the tested seeds had germination viability. The degree of organic fertilizer decomposition and fertilizer toxicity were judged by the seed germination rate. Figure 1 The seed germination rate analysis showed that the germination rate of compost in each treatment gradually increased with the decomposition process. The germination rate of the fifth sample (after decomposition) reached more than 85%, indicating that the compost in each treatment had low toxicity and met the national standard requirements.

[0046] (II) Analysis of pH and conductivity changes and their causes during composting The changes in pH and conductivity during different composting periods are shown in the figure. Figure 2 , where (a) is the pH value and (b) is the conductivity.

[0047] The pH values ​​of the compost in all treatments showed a continuous downward trend. Initially, they were all in the slightly alkaline range of 8.5-9.2. By the end of composting, the pH values ​​of all treatments had dropped back to 7.4-8.4, stabilizing and meeting national standards. The main reason for the pH decrease was that the ammonification of cow manure in the early stage released ammonia nitrogen, maintaining a high pH level. After entering the cooling and maturation stage, the organic acids produced by microbial decomposition of organic matter, the loss of ammonia volatilization, and the buffering effect of humus all contributed to the gradual decrease in pH. This process also reflects the natural succession of the composting system towards the maturation stage. Among them, the grape vine + cow manure 1:1 group (N1, N2, N3) had the closest final pH to neutral, at 7.42, 7.45, and 7.66 respectively, which is in the slightly alkaline range. This indicates that its composting system has a stronger buffering capacity, a higher degree of maturation, and the products are more easily absorbed and utilized by crops. Furthermore, this organic fertilizer can help reduce the pH value of slightly alkaline soils in Ningxia, and has a certain promoting effect on alleviating soil salinization.

[0048] The EC (electrical conductivity) value of the N1 treatment group increased from approximately 4.58 initially to 8.78 in the second sampling, and then gradually decreased to 3.88 in the fifth sampling. The EC value of the N2 treatment group increased from approximately 3.65 initially to approximately 9.79 in the second sampling, and then decreased to 4.10 in the fifth sampling. The EC value of the N3 treatment group increased from approximately 3.20 initially to approximately 9.22 in the second sampling, and then decreased to approximately 3.40 in the fifth sampling. In N1, the MC composting agent decomposes lignin and other recalcitrant components, initially releasing small molecule salts that cause the EC to rise. Later, after composting, the salts are lost or fixed, and the EC falls back. In N2, the T composting agent rapidly decomposes organic matter, initially releasing ammonium nitrogen and other salts that cause a sharp rise in EC. Later, the humus fixes nutrients and the loss of salts increases, resulting in a more significant decrease in EC. N3 relies on indigenous microorganisms. Initially, salt release causes the EC to rise, but later, the nitrogen fixation capacity is weak, and the continuous loss of salts leads to a large decrease in EC.

[0049] The EC value of the N4 treatment group increased from approximately 5.02 initially to approximately 11.94 in the second sampling, and then decreased to approximately 5.56 in the fifth sampling. The EC value of the N5 treatment group increased from approximately 5.17 initially to approximately 11.28 in the second sampling, and then decreased to approximately 4.37 in the fifth sampling. The EC value of the N6 treatment group increased from approximately 5.01 initially to approximately 12.54 in the second sampling, and then decreased to approximately 8.17 in the fifth sampling. The high proportion of cow manure in the N4 treatment group, combined with MC composting inoculant, resulted in a high EC peak due to the initial release of a large amount of salts from decomposition. In the later stages, enhanced microbial synthesis fixed salts, and the EC gradually decreased. The high proportion of cow manure in the N5 treatment group, combined with T composting inoculant, resulted in a significant increase in EC initially. In the later stages, the inoculant promoted humus synthesis and fixed nutrients, and the EC decreased to a lower level. The high proportion of cow manure in the N6 treatment group led to the highest EC peak due to the initial salt release by indigenous microorganisms. In the later stages, insufficient nutrient fixation resulted in a significant decrease in EC.

[0050] The EC value of the N7 treatment group increased from approximately 10.19 initially to approximately 24.43 in the second sampling, and then decreased to approximately 7.76 in the fifth sampling. The EC value of the N8 treatment group increased from approximately 9.84 initially to approximately 22.53 in the second sampling, and then decreased to approximately 7.71 in the fifth sampling. The EC value of the N9 treatment group increased from approximately 10.63 initially to approximately 23.80 in the second sampling, and then decreased to approximately 8.17 in the fifth sampling. The N7 treatment group had more easily degradable components in its pure cow manure. The initial salt release of the MC composting agent led to the highest EC peak, while the lack of carbon source in the later stage reduced microbial activity, and the EC decreased with the loss of salt. The N8 treatment group had good aeration of pure cow manure. The rapid salt release of the T composting agent in the early stage pushed up the EC, but the synthesis weakened and salt was lost in the later stage, resulting in a significant decrease in EC. The N9 treatment group had rich organic matter in its pure cow manure. The initial salt release of indigenous microorganisms led to the highest EC peak, while the lack of substrate in the later stage caused the largest decrease in EC.

[0051] In summary, the EC values ​​of the nine treatments generally showed a trend of "initial increase → peak in the middle stage → decline in the later stage," which is consistent with the stage characteristics of salt release during the thermophilic period of composting, salt loss and fixation during the cooling period, and salt stability during the maturation period. In terms of substrate ratio, the pure cow manure treatment had the highest EC peak, followed by the 1:2 ratio of grape branches + cow manure, while the 1:1 ratio of grape branches + cow manure had the lowest peak due to the carbon-nitrogen ratio balance. In terms of microbial agent type, the T-type maturation agent promoted humus synthesis in the later stage, resulting in a more significant decline in EC. The MC-type maturation agent showed a moderate decline, while the blank control (N3, N6, N9) showed the largest decline due to its weak fixation capacity. Among them, N5, N2, and N4 performed best. N5 had a moderate peak value and then dropped to a low level. T composting agent had outstanding decomposition and fixation capabilities, and the product had the best salt stability and composting degree. N2 had a low peak value and a stable drop. The 1:1 ratio of grape branches and cow manure with T composting agent synergistically prevented excessive salt accumulation and the product had strong safety. N4 had a moderate peak value and a gradual drop. MC composting agent was suitable for high lignin substrates and had good composting stability.

[0052] (III) Analysis of Changes and Causes of Available Nutrients During Composting Changes in available nutrient content during composting are shown in the figure. Figure 3 Among them, (a) is alkaline hydrolyzable nitrogen, (b) is available phosphorus, and (c) is readily available potassium.

[0053] from Figure 3As can be seen, there are significant differences in the changes of alkaline nitrogen content in each treatment: Group N1 gradually increased from an initial value of approximately 638.17 mg / kg, reaching a peak of approximately 1040.67 mg / kg in the fourth sampling, and then dropped back to approximately 882.00 mg / kg, showing an overall trend of "increase-peak-decline"; Group N2 started at approximately 641.67 mg / kg, and reached approximately 844.67 mg / kg in the fifth sampling; Group N3 started at approximately 565.83 mg / kg, rose to approximately 842.33 mg / kg in the second sampling, and then dropped sharply to approximately 658.00 mg / kg, reaching approximately 791 mg / kg in the fifth sampling, showing a large fluctuation range. In the grape vine + cow manure 1:2 group, the N4 group initially had a concentration of approximately 726.83 mg / kg, peaked at approximately 1178.33 mg / kg in the fourth sampling, and then dropped to approximately 907.67 mg / kg, which was the highest peak among all treatments. The N5 group initially had a concentration of approximately 648.67 mg / kg, rose to approximately 1096.67 mg / kg in the fourth sampling, and then dropped to approximately 910.00 mg / kg in the fifth sampling, showing a clear overall upward trend. The N6 group initially had a concentration of approximately 561.17 mg / kg, rose to approximately 961.33 mg / kg in the fifth sampling, and the increase was relatively gradual. In the pure cow manure groups, the N7 group initially had a concentration of approximately 642.83 mg / kg, which rose to approximately 1003.33 mg / kg in the second sampling and remained stable. In the fifth sampling, it dropped back to approximately 991.67 mg / kg, showing relatively stable changes throughout the fermentation cycle. The N8 group initially had a concentration of approximately 710.50 mg / kg, which stabilized at approximately 947.33 mg / kg in the fifth sampling. The N9 group initially had a concentration of approximately 696.50 mg / kg, which dropped back to approximately 844.67 mg / kg in the fifth sampling, showing a moderate overall increase.

[0054] In summary, the overall trend of alkaline nitrogen hydrolysis is mainly driven by substrate ratio, exogenous microbial agents, and composting stage: the carbon-nitrogen ratio of the grape vine + cow manure 1:2 group (N4, N5, N6) is closer to the optimal range of microbial decomposition, with stronger microbial activity and nitrogen mineralization, and higher alkaline nitrogen content. The effects are less pronounced in the grape vine + cow manure 1:1 group with higher carbon and the pure cow manure group with higher nitrogen. MC composting agents (N1, N4, N7) can efficiently degrade lignin and cellulose, while T composting agents (N2, N5, N8) can promote the composting of organic matter, both of which accelerate nitrogen mineralization. Among the best-performing N4, N7, and N5, N4 (grape twigs + cow manure 1:2 + MC composting microbial agent) has the highest nitrogen mineralization efficiency and the highest peak value of alkaline nitrogen due to its suitable carbon-nitrogen ratio and the synergistic decomposition of lignin and nitrogen-containing organic matter by the microbial agents and strains. N7 (pure cow manure + MC composting microbial agent) relies on the high initial nitrogen content of pure cow manure, and the microbial agent effectively decomposes complex organic matter and reduces nitrogen loss, resulting in a stable and high nitrogen content throughout the process. N5 (grape twigs + cow manure 1:2 + T composting microbial agent) can rapidly increase and maintain a high level of alkaline nitrogen due to its suitable carbon-nitrogen ratio and the synergistic effect of multiple strains of microbial agents.

[0055] from Figure 3 As can be seen, the trends in available phosphorus content across treatments were generally consistent: Group N1 initially had approximately 366.46 mg / kg, initially increasing before decreasing to approximately 398.01 mg / kg; Group N2 initially had approximately 222.02 mg / kg, decreasing to approximately 383.49 mg / kg; Group N3 initially had approximately 317.70 mg / kg, decreasing to approximately 354.36 mg / kg; Group N4 initially had approximately 374.92 mg / kg, decreasing to approximately 491.00 mg / kg; Group N5 initially had approximately 341.37 mg / kg, decreasing to approximately 452.68 mg / kg; Group N6 initially had approximately 316.93 mg / kg, decreasing to approximately 404.73 mg / kg; Group N7 initially had approximately 463.59 mg / kg, decreasing to approximately 377.15 mg / kg; Group N8 initially had approximately 432.78 mg / kg, decreasing to approximately 355.47 mg / kg; and Group N9 initially had approximately 455.89 mg / kg. mg / kg to a final dose of approximately 314.37 mg / kg.

[0056] All nine treatments showed a trend of "initial increase or slow decrease → mid-term sharp drop → late-term rebound" in available phosphorus, which highly coincided with the stage characteristics of phosphorus release during the thermophilic period of composting, phosphorus consumption and fixation during the cooling period, and phosphorus re-release during the maturation period. The 1:2 ratio of grape twigs and cow manure showed the highest peak available phosphorus, followed by the 1:1 ratio. The pure cow manure treatment had a smaller rebound in the later stage due to insufficient carbon source. The blank controls (N3, N6, N9) relied solely on indigenous microorganisms, and their phosphorus regulation efficiency was weaker than that of the microbial agent treatments. Among them, N1, N3, and N4 performed best. The N1 treatment group benefited from the balanced carbon-nitrogen ratio and the continuous phosphorus release and efficient phosphorus dissolution of the microbial agent, resulting in outstanding retention efficiency. In the N3 treatment group, the indigenous microorganisms, under the balanced ratio, brought phosphorus release and fixation to a balance, maintaining a high level. In the N4 treatment group, the high phosphorus source combined with the decomposition and phosphorus dissolution of the microbial agent resulted in a stable rebound in available phosphorus.

[0057] from Figure 3As can be seen, the available potassium content in each treatment generally showed a trend of "continuous increase in the initial stage, high level in the middle stage, and decline in the later stage": In the grape vine + cow manure 1:1 group, N1 initially was about 6.46 g / kg, which increased continuously, remained at a high level for a short period, and then declined to about 9.96 g / kg; N2 initially was about 7.06 g / kg, which increased rapidly and remained at a high level before declining to about 9.66 g / kg; N3 initially was about 3.67 g / kg, which increased first and then declined sharply to about 6.36 g / kg. In the grape vine + cow manure 1:2 group, N4 initially was about 9.36 g / kg, which increased rapidly and then declined to about 9.16 g / kg; N5 initially was about 10.46 g / kg, which increased continuously and then declined to about 8.66 g / kg; N6 initially was about 8.76 g / kg, which increased first and then declined to about 8.56 g / kg. In the pure cow dung group, N7 initially had an amount of about 8.15 g / kg, which rose rapidly and then fell back to about 15.65 g / kg; N8 initially had an amount of about 12.05 g / kg, which rose rapidly and remained at a high level before falling back to about 11.26 g / kg; and N9 initially had an amount of about 9.36 g / kg, which rose rapidly and then fell back to about 12.95 g / kg.

[0058] The overall trend shows that in the early stage of composting, microbial decomposition releases bound potassium, which, combined with the concentration effect of water loss, causes it to rise rapidly. In the middle stage, the compost pile stabilizes and maintains a high concentration, while in the later stage, it falls back due to water loss and enhanced microbial fixation. In terms of substrate ratio, the grape twig + cow manure 1:2 ratio and the pure cow manure treatment have a higher peak value of available potassium than the grape twig + cow manure 1:1 ratio treatment due to the higher proportion of cow manure and richer initial potassium. In terms of inoculant type, MC composting inoculant has a stronger potassium fixation ability and better retention efficiency in the later stage, while the blank control (N3, N6, N9) relies on indigenous microorganisms and has more obvious potassium loss.

[0059] (iv) Analysis of the changes and causes of total nutrient content during composting Changes in total nutrient content during composting are shown in the figure. Figure 4 , where (a) is total nitrogen, (b) is total phosphorus, and (c) is organic matter.

[0060] from Figure 4As can be seen, under different treatments, the total nitrogen content showed a trend of "increasing-decreasing-rebounding-slightly decreasing / stabilizing," and was significantly affected by the substrate ratio and the type of microbial agent: In the grape vine + cow manure 1:1 group, N1 (initial approx. 9.10 g / kg, final approx. 10.34 g / kg), N2 (initial approx. 9.13 g / kg, final approx. 10.17 g / kg), and N3 (initial approx. 9.07 g / kg, final approx. 10.79 g / kg) all showed an initial increase in nitrogen release, a decrease in ammonia volatilization loss in the middle stage, and a rebound in nitrogen fixation in the later stage. Among them, N1 relied on MC composting microbial agent to decompose nitrogen release and fix nitrogen, N2 relied on T composting microbial agent to promote humus synthesis and nitrogen fixation, and N3 relied on indigenous microorganisms, with a weaker regulation efficiency than the microbial agent group; In the grape vine + cow manure 1:2 group, N4 (initial approx. 8.46 g / kg, final approx. 10.61 g / kg), N5 (initial approx. 9.48 g / kg, final approx. 10.61 g / kg), and N5 (initial approx. 9.48 g / kg, final approx. 10.61 g / kg), and N3 (initial approx. 9.48 g / kg, final approx. 10.79 g / kg) all showed a trend of "increasing-decreasing-rebounding-slightly decreasing / stabilizing," and were significantly affected by the substrate ratio and the type of microbial agent: In the pure cow manure group, N7 (initial approx. 6.79 g / kg, final approx. 10.37 g / kg), N6 (initial approx. 9.75 g / kg, final approx. 9.91 g / kg), benefited from the rich nitrogen source of the high cow manure ratio. In the early stage, nitrogen release increased, loss decreased in the middle stage, and nitrogen fixation rebounded in the later stage. N4 and N5 were better regulated by MC composting agent and T composting agent, respectively. N6 relied on indigenous microorganisms and had low efficiency. In the pure cow manure group, N7 (initial approx. 6.79 g / kg, final approx. 9.09 g / kg), N8 (initial approx. 9.45 g / kg, final approx. 8.90 g / kg), and N9 (initial approx. 9.18 g / kg, final approx. 8.68 g / kg) all showed an increase in nitrogen release in the early stage, a decrease in loss in the middle stage, and a rebound in nitrogen fixation in the later stage. N7 and N8 were regulated by MC composting agent and T composting agent, respectively. N9 relied on indigenous microorganisms. The pure cow manure without aseptic agent regulation resulted in an overall low final value.

[0061] All nine treatments showed a trend of "initial increase, mid-term decrease, and late-term rebound" in total nitrogen content, which highly coincided with the stage characteristics of nitrogen release during the thermophilic period of composting, ammonia volatilization loss during the cooling period, and nitrogen fixation during the maturation period. In terms of substrate ratio, the 1:2 ratio of grape branches and cow manure had a more abundant nitrogen source, and the total nitrogen content was higher than that of the 1:1 ratio. The pure cow manure treatment had weaker nitrogen stability in the later stage due to insufficient carbon source than the treatment with added grape branches. In terms of microbial agent type, the T-type maturation agent had stronger humus synthesis and nitrogen fixation capabilities in the later stage, and the total nitrogen rebound was greater. The MC-type maturation agent had a more gradual nitrogen release and fixation. The nitrogen regulation efficiency of the blank control was weaker than that of the agent treatment. Among them, N2, N4, and N5 performed best. N2 (1:1 (grape vines + cow manure) + T composting agent) had a final total nitrogen of about 10.17 g / kg. The carbon-nitrogen ratio balance and the synergistic effect of the agent resulted in high nitrogen retention efficiency and significant recovery in the later stage. N4 (1:2 (grape vines + cow manure) + MC composting agent) had a final total nitrogen of about 10.61 g / kg. The high nitrogen source combined with the decomposition and synthesis of the agent resulted in good nitrogen stability. N5 (1:2 (grape vines + cow manure) + T composting agent) had a final total nitrogen of about 10.37 g / kg. The agent had both efficient nitrogen release and nitrogen fixation, and excellent nitrogen retention and stability.

[0062] from Figure 4As can be seen, the total phosphorus content of most treatments showed a trend of "a slight decrease in the early stage and a continuous increase in the later stage". In the grape twig + cow manure 1:1 group, N1 (initial about 4.63 g / kg, final about 6.77 g / kg) decreased slightly due to the consumption of soluble phosphorus by microorganisms in the early stage of MC composting agent, and increased in the later stage due to the concentration effect and phosphorus release of the agent; N2 (initial about 4.77 g / kg, final about 7.05 g / kg) decreased due to the decrease in phosphorus consumption by microorganisms in the early stage of T composting agent, and increased significantly due to the phosphorus solubility and concentration effect in the later stage; N3 (initial about 4.78 g / kg, final about 5.76 g / kg) depended on indigenous microorganisms, and increased less than that of the agent group due to natural concentration in the later stage. In the grape vine + cow manure 1:2 group, N4 (initially about 5.76 g / kg, ultimately about 8.68 g / kg) relied on the high cow manure phosphorus source, and the phosphorus release and concentration effect of MC composting agent in the later stage caused its greatest increase; N5 (initially about 5.40 g / kg, ultimately about 7.69 g / kg) was significantly increased in total phosphorus due to the synergistic phosphorus dissolution of T composting agent in the later stage; N6 (initially about 5.17 g / kg, ultimately about 6.53 g / kg) relied only on the concentration effect in the later stage, and its increase was relatively small. In the pure cow dung group, N7 (initially about 6.05 g / kg, ultimately about 6.43 g / kg) had weak microbial activity due to insufficient carbon source, and the phosphorus release in the later stage of MC composting agent caused the increase to be slow; N8 (initially about 5.89 g / kg, ultimately about 6.94 g / kg) had a significant increase in total phosphorus due to the efficient phosphorus dissolution in the later stage of T composting agent; N9 (initially about 5.71 g / kg, ultimately about 5.74 g / kg) had no significant increase in total phosphorus due to natural concentration in the later stage.

[0063] The total phosphorus content of the nine treatments mostly showed a trend of "slight decrease in the early stage, continuous increase in the middle stage, and rapid increase in the later stage". In the early stage, the total phosphorus content was slightly reduced due to the consumption of soluble phosphorus by microbial proliferation, and the release of insoluble phosphorus by microbial decomposition of organic matter drove the increase in total phosphorus. In terms of substrate ratio, the treatment with grape twigs + cow manure in a 1:2 ratio had a higher proportion of cow manure and was richer in initial phosphorus. Combined with the carbon source of grape twigs to enhance microbial activity, the total phosphorus content increased the most. The treatment with grape twigs + cow manure in a 1:1 ratio was the second highest. The treatment with pure cow manure had the smallest increase due to insufficient carbon source and weak microbial activity. In terms of microbial agent type, the T composting agent significantly increased the total phosphorus content by means of the phosphorus-solubilizing effect of Bacillus subtilis and Trichoderma viride. The MC composting agent had a moderate phosphorus-solubilizing effect. The blank control (N3, N6, N9) only relied on natural concentration and had the smallest increase. Among them, N4, N5, and N2 performed best. N4 (1:2 (grape twigs + cow manure) + MC composting agent) had the highest final total phosphorus content, thanks to the high initial phosphorus source, the release of insoluble phosphorus from the decomposition of the agent, and the concentration effect of the pile. N5 (1:2 (grape twigs + cow manure) + T composting agent) had a high phosphorus source and the agent worked together to dissolve phosphorus, and the superimposed concentration effect increased the phosphorus concentration. N2 (1:1 (grape twigs + cow manure) + T composting agent) had a balanced carbon-nitrogen ratio, and the agent had multiple microbial communities working together to decompose and dissolve phosphorus, resulting in high total phosphorus retention efficiency.

[0064] from Figure 4 As can be seen, the initial organic matter content of the N1 treatment group was 615.81 g / kg, and the final content was 343.27 g / kg, showing a slow downward trend. The initial organic matter content of the N2 treatment group was approximately 567.30 g / kg, and the final content was 379.65 g / kg, showing a rapid decline in the early stage followed by a plateau. The initial organic matter content of the N3 treatment group was approximately 548.09 g / kg, and the final content was approximately 315.75 g / kg, showing a continuous decline with slight fluctuations in the middle stage. All three treatments (N1, N2, and N3) used a 1:1 ratio of grapevine branches and cow manure. Among them, N1 decomposed recalcitrant components due to the MC composting agent, resulting in moderate decomposition in the early stage and a slowdown in the later stage. N2 rapidly decomposed easily degradable substances in the early stage with the T composting agent, and the decomposition and synthesis tended to be in equilibrium in the later stage, resulting in high retention efficiency. N3 did not have any exogenous inoculants added and relied on indigenous microorganisms, resulting in a weaker decomposition intensity than the inoculant treatment, but a brief accumulation of organic matter occurred in the middle stage.

[0065] The initial organic matter content of N4 was approximately 482.03 g / kg, and the final content was approximately 357.21 g / kg, showing an initial decrease followed by stabilization and a slight increase in the later stages. The initial organic matter content of N5 was approximately 527.16 g / kg, and the final content was approximately 417.41 g / kg, showing an initial decrease followed by a slow increase and maintaining a high level. The initial organic matter content of N6 was approximately 490.41 g / kg, and the final content was approximately 321.74 g / kg, showing a continuous decrease with a narrowing rate of decline in the later stages. N4, N5, and N6 all used a 1:2 mixture of grapevine branches and cow manure, providing abundant readily biodegradable nitrogen and substrate. N4 saw a slight increase in content in the later stages due to the decomposition of recalcitrant components by the MC composting agent. N5 benefited from the synergistic effect of the T composting agent, which promoted humus synthesis, resulting in an increase in organic matter content that remained at a high level. N6, lacking exogenous inoculants and relying on indigenous microorganisms, had a lower content in the later stages.

[0066] The initial organic matter content of N7 was approximately 360.12 g / kg, and the final organic matter content was approximately 268.25 g / kg, showing a gradual decrease throughout the process. The initial organic matter content of N8 was approximately 349.85 g / kg, and the final organic matter content was approximately 213.61 g / kg, showing the largest decrease throughout the process. The initial organic matter content of N9 was approximately 351.86 g / kg, and the final organic matter content was approximately 243.49 g / kg, showing a rapid decrease followed by a slowdown. N7, N8, and N9 are all pure cow manure compost, with a high proportion of easily degradable organic matter. N7, with the addition of MC composting microbial agent, decomposed slowly overall because the agent focuses on decomposing recalcitrant lignin. N9, without exogenous microbial agents, decomposed rapidly in the early stages due to the presence of native microorganisms, but the rate decreased later due to insufficient substrate.

[0067] The three treatments with the best performance were N5, N2, and N4, in descending order. Among them, N5 benefited from the sufficient nitrogen source provided by the high proportion of cow manure, and the combination with T composting agent effectively promoted humus synthesis. Not only did it have the highest organic matter content, but it also showed a significant rebound in the later stage, resulting in the best degree of humification. N2 used a 1:1 substrate ratio of grape branches and cow manure to achieve a dynamic balance of carbon and nitrogen ratio. Combined with the synergistic effect of multiple microbial groups of T composting agent, it not only ensured the composting effect but also improved the organic matter retention efficiency, keeping its content stable in the later stage. N4 achieved its best results through the combination of a high proportion of cow manure and MC composting agent. In addition, all nine treatments followed the natural succession pattern of composting, showing a trend of "rapid decomposition in the early stage - slowing down in the middle stage - stabilizing / rebounding in the later stage". This phenomenon was mainly regulated by the substrate ratio and the type of microbial agent. The addition of grape branches can increase the initial organic matter content and carbon-nitrogen ratio, while the proportion of cow manure directly affects the initial organic matter decomposition rate. The T composting agent focuses more on the balance between decomposition and synthesis, the MC composting agent is good at degrading recalcitrant components, while the blank control relies on indigenous microorganisms, and its decomposition efficiency is lower than that of the microbial agent treatment.

[0068] (V) The effects of different treatments on the nutrient content of organic fertilizer were investigated using a two-way ANOVA. The two-factor analysis chart of the physicochemical indicators of the last sample of compost is shown below. Figure 5 .

[0069] from Figure 5 The results show that the raw material ratio and the type of microbial agent have extremely significant effects on the nutrient content of compost (P<0.001 or P<0.01). Except for alkaline nitrogen, the interaction effects of the other indicators are all extremely significant, indicating that the effect of the microbial agent is highly dependent on the raw material ratio. Regarding the core fertilizer efficiency indicators, the 2:1 ratio (cow manure: grapevine branches) performed best. Under the same microbial agent conditions, the organic matter, total nitrogen, total phosphorus, and available phosphorus contents of this ratio were significantly higher than those of the 1:1 (cow manure: grapevine branches) and 1:0 (pure cow manure) ratios. The 1:0 (pure cow manure) ratio only showed an advantage in available potassium content, with the lowest values ​​for the other indicators. Inoculation with MC composting microbial agent and T composting microbial agent significantly increased the contents of organic matter, total phosphorus, and available phosphorus. The T composting microbial agent was more conducive to organic matter retention, but both agents reduced the available potassium content, with the blank control group having the highest available potassium content. The interaction patterns of alkaline nitrogen content differed. In the blank control group, the ratio effect was extremely significant, showing 2:1 > 1:0 > 1:1. However, after inoculation with microbial agents, the differences between different ratios were not significant. In summary, the optimal combination for preparing high-quality organic fertilizer is MC composting agent or T composting agent with a 2:1 ratio of raw materials (cow manure: grape branches), which can provide a theoretical basis for the practical application of grape branch and cow manure compost.

[0070] (vi) The effects of different composting treatments on microorganisms The stacked bar chart of the relative abundance of microbial communities at the phylum level in the last sampling of compost is shown below. Figure 6 Among them, (a) is a stacked bar chart of the relative abundance of bacterial community phyla at different ratios of grape vines and cow manure, and (b) is a stacked bar chart of the relative abundance of fungal community phyla at different ratios of grape vines and cow manure.

[0071] See the ASV species and PCA diagram of the microbial community from the final sampling of compost microorganisms. Figure 7 Among them, (a) shows the common species ASVs of soil bacteria under different ratios of grape twigs and cow manure, (b) shows the common species ASVs of soil fungi under different ratios of grape twigs and cow manure, (c) shows the PCA diagram of bacterial community under different ratios of grape twigs and cow manure, and (d) shows the PCA diagram of fungal community under different ratios of grape twigs and cow manure.

[0072] See the microbial community alpha abundance diagram. Figure 8 Among them, (a) is the alpha abundance of bacterial community under different ratios of grape branches and cow manure, and (b) is the alpha abundance of fungal community under different ratios of grape branches and cow manure.

[0073] By using 16S high-throughput absolute and relative quantification techniques to comprehensively analyze the microbial community data of 27 organic fertilizer samples (9 treatments, 3 replicates per treatment), the microbial community structure of organic fertilizers under different treatments showed rich diversity at the phylum level. Figure 6 Together, they revealed the decisive impact of different ratios of cow manure and grape vine organic fertilizer on the compost microbial community. For example... Figure 6 (a) At the phylum level, the number of bacterial phyla in compost varies under different treatments. At the phylum level, Proteobacteria, Bacteroidetes, and Actinobacteria are the dominant bacterial phyla in compost, such as... Figure 6 (b) Ascomycota are the dominant phylum in fungi. From Figure 7 (a) It can be seen that the number of common ASVs among soil bacteria under different treatments was 86, while the number of species specific to N1-N9 were 4522, 5095, 5345, 5032, 4344, 3583, 3189, 3319, and 2436, respectively. Furthermore, the bacterial abundance was significantly increased in the treatment group that added grape branches for composting. Figure 7 (b) It can be seen that the number of common ASVs in soil fungi under different treatments was 5. The number of unique species among N1-N9 were 168, 141, 169, 195, 156, 111, 127, 160 and 84, respectively. The fungal abundance was significantly increased in the treatment group with grape branches added for composting. Combining the stacked bar chart of relative abundance at the bacterial and fungal phylum levels and the ASV species information, it can be seen that adding grape branches helps to increase the abundance of the microbial community, thereby facilitating composting and improving the situation of a monotonous microbial community.

[0074] Figure 7 PCA analyses in (c) and (d) indicate that the ratio of raw materials is the core factor driving community differentiation. Bacterial PC1 and PC2 explain 47.7% and 24.2% of community variation, respectively, while fungal PC1 and PC2 explain 72.7% and 13.1%, respectively. Samples with different ratios show significant separation along the PC1 axis. Under the same ratio, the inoculant regulation effect is better and more conducive to the fungal community. Bacteria are more complexly affected by both factors, and fungi show a higher degree of aggregation in samples with the same ratio. In conclusion, the ratio of grape vine branches to cow manure is the core factor regulating the microbial community, and the addition of inoculants can effectively improve the microbial community structure.

[0075] Figure 8(a) and (b) show that different compost ratios significantly regulate microbial community diversity. Except for the fungal Goods_coverage index (p=0.18), all diversity indices, including Chao1 and Shannon, had p-values ​​<0.05, indicating significant differences between groups. All indices showed consistent trends: species richness and phylogenetic diversity were highest in the N1-N3 treatments and lowest in the N7-N9 (pure cow manure) treatments. Regarding species evenness (Pielou_e), the evenness of bacterial and fungal communities varied with the compost ratio. In conclusion, the compost ratio is a key factor determining microbial community diversity (quantity, evenness, and evolutionary diversity). The N1-N3 (1:1 ratio) compost, due to its abundant carbon source and microenvironment, resulted in a more stable and richer community. While the pure cow manure compost, with its single fermentation material, limited diversity, resulted in relatively mild competition among dominant species.

[0076] In summary, the ratio of grape vine branches to cow manure is the core factor in regulating soil microbial communities. T composting agent can effectively change the microbial community structure under specific ratios, and bacteria and fungi show significant differences in their responses to the ratio of raw materials and the addition of the agent. These results can provide a theoretical basis for the microbial regulation of grape vine and cow manure compost and the improvement of organic fertilizer quality.

[0077] (vii) Effects of different composting treatments on the physicochemical properties and plant growth of potted grapes. The organic fertilizer prepared through the organic fertilizer preparation experiment was used in a pot experiment. After 3 months of greenhouse pot growth, its physicochemical properties and plant growth were measured and analyzed. Healthy, disease-free grape cuttings of the 'Viony' variety were selected for the potted seedlings, with 3-5 leaves. The basic physicochemical properties of the potting substrate (low nutrient substrate: organic = 100:5) are as follows: pH value: 6.06, electrical conductivity: 109.53 μS / cm, alkaline nitrogen content: 241.791 mg / kg, available phosphorus content: 71.42 mg / kg, available potassium content: 336.660 mg / kg, total nitrogen content: 5.89 g / kg, total phosphorus content: 4.70 g / kg, and organic matter content: 183.05 g / kg.

[0078] As shown in Table 2, the pH values ​​of the potted plants under different treatments did not differ significantly. This is related to the release of alkaline substances such as ammonia, calcium, and magnesium ions during the mineralization process of organic fertilizer (especially cow manure). The overall trend was stable, which is consistent with the suitable growth range for grape seedlings. The electrical conductivity (EC) increased significantly from the initial 109.53 mS / cm to 200-500 μS / cm. The EC values ​​were higher in treatments N4, N5, N6 (grape twigs + cow manure in a 1:2 ratio) and N7, N8, N9 (pure cow manure). This is because the mineralization of organic fertilizer releases a large amount of soluble salts (such as potassium, calcium, magnesium, and ammonium ions), and the high proportion of cow manure treatments resulted in a more sufficient input of mineral elements, which is a reasonable trend.

[0079] Soil-available nitrogen mainly consists of ammonium nitrogen, nitrate nitrogen, and easily hydrolyzable organic nitrogen. Soil-available nitrogen is the part that plants can easily absorb during growth and development. During the three-month growth period of potted plants, the content of soil-available nitrogen changed from the initial 241.791 mg / kg to 142.63~237.13 mg / kg. This is the result of the combined effect of the large amount of nitrogen consumed by the vegetative growth of grape seedlings (leaf expansion, new shoot growth, root system construction) and the replenishment of organic nitrogen mineralization. Among them, the organic nitrogen content of the pure cow manure organic fertilizer treatment group was relatively low, and the content of converted and released soil-available nitrogen was relatively small. The content of soil-available nitrogen decreased significantly under the consumption of plant growth. Although the nitrogen consumption of the treatments with better growth (N4, N5, N1, N2) was more vigorous, the organic nitrogen mineralization rate was increased simultaneously due to the use of high-efficiency inoculants (MC composting inoculant, T composting inoculant) and appropriate compost ratio, thus maintaining a relatively sufficient nitrogen supply. The contents of available phosphorus and readily available potassium increased in the treatments using grape vines mixed with cow manure at a ratio of 1:2 and in the pure cow manure treatment. This is because the release rate of abundant phosphorus and potassium mineralization in cow manure exceeded the absorption rate of the plants, forming a virtuous cycle of "high supply-high consumption-high surplus," which provided a guarantee for root development and the construction of stress resistance. The total nitrogen content decreased overall from the initial 5.89 g / kg due to continuous consumption during the growth and development of the potted plants. The contents of total phosphorus and organic matter remained stable except for some treatments where they increased. This is consistent with the characteristics of nitrogen being easily converted and absorbed or lost in gaseous form, phosphorus being stable and not easily leached, and organic fertilizer continuously inputting organic matter.

[0080] From the perspective of treatment differences, grouped by compost ratio, the 1:1 ratio of grape vine branches + cow manure, due to the high carbon-to-nitrogen ratio of grape vines and slow mineralization rate, results in a relatively mild nutrient release. Combined with MC and T composting agents, this improves the efficiency of organic nitrogen mineralization, allowing the plants to grow steadily in a stable nutrient supply environment and avoiding the risk of "burning" the seedlings. N4, N5, and N6 (1:2 ratio of grape vine branches + cow manure), with an increased proportion of cow manure, provide more abundant mineral elements and organic matter input. Combined with highly efficient microbial agents, the release of fast-acting nutrients is more complete, supporting vigorous vegetative growth. N7, N8, and N9 (pure cow manure), with the richest reserves of nitrogen, phosphorus, potassium, and organic matter, have the highest total nutrient content, providing a continuous and stable nutrient supply to the plants. Grouped by inoculant type, MC composting inoculants (N1, N4, N7) can effectively decompose cellulose and hemicellulose, promote the mineralization of readily available nutrients, and result in relatively high EC and readily available nutrient content; T composting inoculants (N2, N5, N8) focus on optimizing the microbial community and promoting the humification of organic matter, improving the substrate's fertilizer retention capacity and buffering performance, making nutrients less prone to leaching, and more conducive to the continuous growth of plants.

[0081] Table 2 Physicochemical properties of potted seedlings under different organic fertilizer applications Based on comprehensive physicochemical indicators and plant growth, the optimal treatments were N4 (1:2 ratio of grapevine branches to cow manure + MC composting agent) and N5 (1:2 ratio of grapevine branches to cow manure + T composting agent). N4 provides sufficient nitrogen, phosphorus, and potassium through the 1:2 ratio of grapevine branches to cow manure, while the MC composting agent accelerates the mineralization of organic nutrients, maintaining available phosphorus, available potassium, and EC at high levels. Simultaneously, the high organic matter content buffers the increase in EC, preventing salt stress. N5 provides a high potassium source through the 1:2 ratio of grapevine branches to cow manure, while the T composting agent optimizes the microbial community, improving fertilizer retention capacity and nutrient utilization efficiency. EC and pH are within suitable ranges, providing a favorable environment for root development and stress resistance.

[0082] In summary, the treatments (N4, N5, N1, N2) that favored the growth of potted grape seedlings demonstrated that appropriate compost ratios provided abundant nutrient reserves, highly effective microbial agents accelerated nutrient release, and high organic matter content buffered changes in EC and pH, ultimately providing ideal conditions for grape seedling growth. These results indicate that both the proportion of compost materials and the type of microbial agent significantly affected the growth of grape seedlings, and there was a clear interaction between the two.

[0083] In terms of compost material ratios, the grape vine + cow manure 1:2 ratio group (N4, N5, N6) performed best overall, superior to the grape vine + cow manure 1:1 ratio group (N1, N2, N3) and the pure cow manure group (N7, N8, N9). This is because the grape vine + cow manure 1:2 ratio increases nitrogen source supply and facilitates the release of readily available nitrogen, which can quickly meet the nitrogen needs of grape seedlings in the early stages of growth, promoting leaf growth and photosynthesis. On the other hand, the pure cow manure group, due to insufficient carbon source and excessively high nitrogen concentration, is prone to ammonia volatilization and salt accumulation. At the same time, the lack of lignin and other structural substances to support soil aggregate structure results in poor soil aeration and hindered root development, thereby inhibiting seedling growth.

[0084] From the perspective of microbial inoculant types, the addition of MC composting inoculants (N1, N4, N7) and T composting inoculants (N2, N5, N8) were significantly superior to the blank control group (N3, N6, N9) without exogenous bacteria. Bacillus licheniformis, Xanthomonas typhimurium, and Pseudomonas beichengensis in the MC composting inoculant effectively decompose cellulose and lignin, promoting compost maturation, while secreting plant growth hormones (such as IAA), stimulating root development and nutrient absorption, and enhancing plant resistance. Saccharomyces cerevisiae, Bacillus subtilis, and Trichoderma viride in the T composting inoculant efficiently decompose organic matter, release nutrients, and improve soil microecology by competitively inhibiting pathogens. However, in a high-nitrogen environment with pure cow manure, the growth-promoting effect of the T composting inoculant was somewhat weakened, while the MC composting inoculant showed strong adaptability at different proportions.

[0085] Considering plant height, foliage density, and overall health, the three treatments with the best performance were N4 (1:2 (grape vines + cow manure) + MC composting agent), N5 (1:2 (grape vines + cow manure) + T composting agent), and N1 (1:1 (grape vines + cow manure) + MC composting agent). Among these, N4 (1:2 (grape vines + cow manure) + MC composting agent) showed the most outstanding growth. Its core advantages are: the 1:2 compost ratio of grape vines to cow manure provides ample nitrogen to meet the rapid growth needs of grape seedlings; the MC composting agent efficiently decomposes lignin and cellulose, promoting compost decomposition and releasing readily available nutrients; and strains such as Bacillus licheniformis secrete growth hormones, stimulating root development and improving nutrient absorption efficiency. N5 (1:2 (grape vines + cow manure) + T composting agent), through the synergistic effect of Saccharomyces cerevisiae and Trichoderma viride, accelerates organic matter mineralization while inhibiting soil-borne diseases, reducing the risk of seedling infection. N1 (1:1 (grape twigs + cow manure) + MC composting agent) has a carbon-nitrogen ratio of 1:1 that is closer to the ideal composting conditions, resulting in a more stable composting process, longer nutrient release, and higher activity of MC composting agent in medium nitrogen environment. It can effectively regulate soil pH and EC value, avoid salt stress, and enable seedlings to maintain stable growth in long-term pot trials.

[0086] The above results indicate that a 1:2 compost ratio of grape branches and cow manure, combined with MC or T composting inoculants, can significantly improve the growth performance of grape seedlings and is a relatively ideal organic fertilizer application scheme. However, pure cow manure without exogenous inoculants can easily lead to soil environmental deterioration and inhibit seedling growth, so it should be used with caution or combined with highly effective inoculants.

[0087] Table 3 Core Morphological Indicators of Grape Seedlings Based on the extensive grape cultivation area and high yield of winter-pruned branches in the eastern foothills of the Helan Mountains in Ningxia, composting grape branches with cow manure has significant advantages over cow manure and other highly lignified branches (such as poplar and apple tree branches). First, the cost advantage of on-site resource utilization is prominent: winter-pruned branches in Ningxia's wine grape producing areas can yield hundreds of kilograms per mu (approximately 0.16 acres), are easy to collect and transport, and can be directly used for composting; while other lignified branches lack large-scale production locally and require long-distance transportation, increasing economic and carbon emission costs. Second, the inherent characteristics of grape branches are more conducive to rapid decomposition: as a vine, its fiber structure is relatively loose, its lignin content is low, and its branch diameter is small (mostly less than 1-2 cm), allowing it to be mixed with cow manure without complex crushing; in contrast, the hardwood branches of poplar, willow, and other hardwood trees are coarse, dense, and require high energy consumption for crushing, and the pores of the compost pile are prone to collapse, inducing anaerobic digestion and prolonging the decomposition period. In summary, based on the resource endowment and production conditions of the Ningxia production area, composting cow manure and grape branches is superior to combinations with other lignified branches in terms of raw material availability, decomposition efficiency, and ecological synergy.

[0088] In the composting process, the combination of cow manure and grapevines is superior to that of chicken manure and grapevines in terms of material ratio compatibility, physical structure, and ecological safety. Regarding the proportion of composting materials, cow manure, being nitrogen-rich, can achieve nutrient balance when mixed with carbon-rich grapevines in an appropriate ratio, promoting efficient microbial metabolism. Chicken manure, however, has a high nitrogen content, and even reducing its proportion in the mixture can easily lead to nitrogen excess, resulting in ammonia volatilization, nitrogen loss, and foul odor. In terms of physical structure, cow manure is loose and has a moderate moisture content, forming a good granular structure when mixed with the hard grapevines, maintaining aeration and facilitating aerobic fermentation. Chicken manure, on the other hand, is dense and has a high moisture content, easily forming crusts and producing harmful gases such as hydrogen sulfide when mixed with the vines. Furthermore, cow manure is rich in cellulose-decomposing bacteria, which can synergistically degrade the lignocellulose in grapevines, improving composting efficiency and quality. Therefore, composting cow manure and grapevines is a more sustainable and environmentally friendly method of organic fertilizer production.

[0089] 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. An organic fertilizer, characterized in that, The raw materials include grapevines, cow dung, and microbial inoculants; The mass ratio of grapevines to cow dung is 1:(1~2).

2. The organic fertilizer according to claim 1, characterized in that, The amount of the microbial inoculant added is 0.50~0.54% of the total mass of grape vines and cow manure.

3. The organic fertilizer according to claim 2, characterized in that, The microbial agents include MC composting agents or T composting agents; And / or, the amount of the MC composting agent added is 0.50% of the total mass of grapevines and cow manure; And / or, the amount of the T composting agent added is 0.54% of the total mass of grape vines and cow manure.

4. The organic fertilizer according to claim 3, characterized in that, The main components of the MC composting agent include Bacillus licheniformis, Pseudomonas beichengensis, and Bacillus pantothenica. And / or, the main components of the T composting agent include Saccharomyces cerevisiae, Trichoderma viride, and Bacillus subtilis.

5. A method for preparing organic fertilizer through co-fermentation of grape vines, cow dung, and microbial inoculants, characterized in that... Includes the following steps: Grape branches are crushed and mixed with cow manure at a mass ratio of 1:(1~2), and microbial agents are added for composting to obtain the organic fertilizer.

6. The method according to claim 5, characterized in that, The length of the crushed grape branches is 0.01~1cm.

7. The method according to claim 5, characterized in that, The parameters for the composting include: turning frequency of 3-5 days / time and moisture content of the compost pile of 50-65%.

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