A compound microbial agent for reducing nitrogen loss in compost and a preparation method and use thereof

Abstract

The present application belongs to the field of waste reuse and environmental pollution control in circular agriculture, and relates to a compound microbial agent for reducing nitrogen loss in compost, a preparation method and use thereof. The compound microbial agent is mixed by Bacillus amyloliquefaciens, Ochrobactrum and Penicillium chrysogenum, and the Bacillus amyloliquefaciens, Ochrobactrum and Penicillium chrysogenum are preserved in the China General Microbiological Culture Collection Center, with the preservation number of Bacillus amyloliquefaciens being CGMCC NO.27935, the preservation number of Ochrobactrum being CGMCC NO.27932, and the preservation number of Penicillium chrysogenum being CGMCC NO.27934. The compound microbial agent provided by the present application is used for compost fermentation, which shortens the preparation time of livestock and poultry manure organic fertilizer, reduces the loss of nitrogen in the form of ammonia and nitrous oxide in the compost, improves the fertilizer efficiency of the organic fertilizer, and promotes the composting of the product.

Description

Technical Field

[0001] This invention belongs to the field of waste recycling and environmental pollution control technology in circular agriculture, and relates to a compound microbial agent for reducing nitrogen loss in composting, its preparation method, and its uses. Background Technology

[0002] With the continuous improvement of people's living standards and the increasing demand for fruits, vegetables, meat, eggs, and other commodities, agriculture and large-scale livestock and poultry farming have developed rapidly, resulting in a large amount of agricultural organic waste such as livestock and poultry manure and straw. Fertilization is the main utilization method for agricultural organic waste. Aerobic composting of crop-degradable organic solid waste enables its utilization as fertilizer and is widely used in the treatment of livestock and poultry manure. However, some engineering practices in recent years have shown that when various organic wastes such as human and animal manure, straw, and vegetable waste are mixed and aerobic fermented to convert them into organic fertilizer products, the complex physicochemical properties and existing forms of the raw materials themselves limit the co-processing of these organic materials. This often results in unstable product quality and significant nitrogen emission losses, making it difficult to fully and quickly dispose of organic waste.

[0003] The main factors affecting the loss of NH3 and nitrogen during composting are the composting process and the composting raw materials. The amount of ammonia volatilization during composting can reach 20% to 70% of the total nitrogen loss. Most of the NH3 diffuses into the atmosphere, causing malodor pollution and reducing the quality of compost. Currently, there are two main approaches to control nitrogen loss in composting: one is to change the process conditions, that is, to reduce gas emissions by controlling the process conditions, including improving ventilation time, material density, turning frequency, moisture content, C / N ratio, etc. This method has a significant effect on nitrogen control, but the operation is cumbersome; the other is to introduce additives into the compost, that is, to add physical adsorbents (wood chips, straw, leaves, fly ash, shredded tires, activated carbon, biochar, peat, zeolite, bentonite, sepiolite, etc.), chemical reagents (superphosphate, ferrous sulfate, dilute sulfuric acid, dilute phosphoric acid, ferric nitrate, ferric chloride, magnesium salts, etc.) and microbial agents (Streptomyces flavus, EM agents, etc.). The potential of this strategy in controlling nitrogen loss in composting has been confirmed by many scholars in experiments, but many studies have also pointed out that the effect of nitrogen loss control in actual composting using additives still varies greatly. The reasons can be summarized as follows: (1) The composition of compost materials is complex, and the physicochemical properties of different materials are very different. For example, unlike livestock and poultry manure and sewage treatment plant sludge, composting with kitchen waste (leftover food mainly composed of starch) as the main raw material is prone to producing acidic substances. Although its nitrogen loss is relatively small, the release of volatile fatty acids is very large, which often causes foul odor or unpleasant smell. However, if alkaline additives (lime, fly ash, etc.) are added to neutralize the acidity, there are very high requirements for the type of additive, the timing of addition, and the amount of addition. Otherwise, it is difficult to achieve the expected effect. (2) There are many types of additives with different physicochemical properties, which may not meet the nitrogen loss control requirements of various composting raw materials. For example, existing technologies can effectively control nitrogen loss in compost by introducing 1%–3% sulfur powder into mixed compost of straw, mushroom residue, and sheep manure. However, this large amount of sulfur powder can lead to excessively acidic (pH < 5) and high salinity (conductivity > 10 mS / cm) compost, making it difficult to meet national standards (pH 5.5–8.5). Furthermore, the strong release of sulfur-containing substances during composting causes severe odor. Conversely, introducing a lower amount of sulfur powder (only 0.8%–1%) into mixed compost of sludge or pig manure with straw or sawdust results in excessively high ammonium salt content (≥ 8 g / kg) and high salinity (conductivity > 7 mS / cm), also failing to meet national standards. Reducing the sulfur powder usage to 0.2% can avoid the problems of low pH and high salinity in compost, but its actual nitrogen retention effect will be inhibited. In addition, sulfur powder is not suitable for composting materials containing kitchen waste, vegetable scraps and branches; (3) Different additives have different uses and functional characteristics, and require higher professional skills from relevant personnel in engineering practice, making it difficult to promote and popularize them.For example, water-soluble iron and aluminum salts such as ferrous sulfate, aluminum sulfate, ferric nitrate, and ferric chloride have highly acidic aqueous solutions, which can absorb NH3 in compost and reduce NH3 volatilization loss. However, they also contain Al. 3+ Fe 2+ Fe 3+ Hydrated hydroxides often react with phosphate in compost to form iron phosphate or aluminum phosphate, which are difficult to be utilized by organisms. Although they play a role in controlling nitrogen loss, they also lead to a sharp decrease in phosphorus utilization, resulting in unsatisfactory actual farmland application effects of compost products (organic fertilizer). Therefore, many scholars suggest that when using water-soluble iron and aluminum salts as additives for composting, operators should make professional and targeted selections based on the basic chemical composition and chemical characteristics of the compost organic materials to achieve good engineering results. (4) The nitrogen loss control of additives in the composting system is also closely related to the engineering conditions of composting. This requires not only to comprehensively determine the type and amount of additives based on the composting method (fully open windrows, semi-open fermentation tanks, sealed fermentation tanks, etc.), gas supply frequency, and organic material mixing ratio, but also to determine the timing of additive addition based on the process of material transformation in the composting process and the emission characteristics of each stage.

[0004] Therefore, compared to physical adsorbents and chemical additives, using microbial agents to control nitrogen loss in composting is considered one of the most promising strategies. This is mainly because composting, or aerobic fermentation composting technology, involves using biomass organic waste (human and animal manure, kitchen waste, agricultural straw, etc.) under aerobic conditions. By artificially controlling environmental temperature, moisture content, carbon-nitrogen ratio, and oxygen supply, microorganisms in the compost rapidly grow and reproduce. Through the action of these microorganisms, macromolecules in the compost are degraded into stable humus, achieving the harmlessness and hygienic treatment of organic waste, thus promoting its fertilizer utilization. Although there are numerous research reports on microbial agents, the materials used in their functional verification tests are still relatively limited, and there are inconsistencies and even contradictions in the results. Since the core of the composting process is controlled by microbial-mediated biochemical reactions, the harmlessness and fertilizer utilization of biomass agricultural organic waste can be achieved by scientifically regulating the composition of the core functional microorganisms in the composting process.

[0005] Therefore, the present invention aims to provide a compound microbial agent that can be used to control nitrogen loss in aerobic composting processes, so as to meet the needs of efficiently realizing the harmlessness and fertilizerization of agricultural biomass organic waste. Summary of the Invention

[0006] The purpose of this invention is to provide a compound microbial agent that reduces nitrogen loss in aerobic composting fermentation substrate.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a compound microbial agent for reducing nitrogen loss in composting, which is composed of Bacillus amyloliquefaciens, Bacillus cereus, and Penicillium chrysogenum. The Bacillus amyloliquefaciens, Bacillus cereus, and Penicillium chrysogenum are all deposited at the China General Microbiological Culture Collection Center (CGMCC). The preservation number of Bacillus amyloliquefaciens is CGMCC NO. 27935, the preservation number of Bacillus cereus is CGMCC NO. 27932, and the preservation number of Penicillium chrysogenum is CGMCC NO. 27934.

[0009] In a second aspect, the present invention provides a method for preparing the aforementioned compound microbial agent, comprising the following steps:

[0010] The Bacillus amyloliquefaciens and the Bacillus licheniformis were respectively inoculated into LB liquid medium and cultured to obtain seed culture of both.

[0011] The Penicillium chrysogenum was inoculated onto PDA solid culture medium and cultured. The resulting bacterial solution was mixed with the two seed solutions mentioned above to obtain the compound bacterial agent.

[0012] Preferably, the seed culture is obtained after culturing at 30°C and 160 rpm for 48 hours.

[0013] Preferably, the effective viable bacteria count of both seed liquids is 10. 8 CFU / mL, the concentration of the bacterial culture is 10. 8 The volume ratio of the seed liquid of Bacillus amyloliquefaciens, the seed liquid of Bacillus cereus, and the bacterial liquid of Penicillium chrysogenum is 1-3:1-3:1-3.

[0014] In a third aspect, the present invention provides the use of the compound microbial agent in composting fermentation.

[0015] Preferably, the compound microbial agent is used to reduce nitrogen loss during composting fermentation.

[0016] In a fourth aspect, the present invention provides a microbial agent comprising the aforementioned compound microbial agent and microbiologically acceptable excipients.

[0017] Preferably, the excipient is a microbial community promoter, which is wheat bran, soybean meal, and Mandels's nutrient solution. The mass ratio of wheat bran to soybean meal is 2-4:1-2; the Mandels's nutrient solution submerges the mixture of wheat bran and soybean meal; the mass ratio of the compound microbial agent to the mixture of wheat bran and soybean meal is 1-4:1.

[0018] In a fifth aspect, the present invention provides a method for composting fermentation using the compound microbial agent, wherein the compound microbial agent is added to the compost material and aerobic fermentation is carried out for 30 to 50 days, and the amount of the compound microbial agent added is 2.5% to 5% based on the dry weight of the compost material, and the moisture content of the compost material is adjusted to 55% to 60% before adding the compound microbial agent.

[0019] In a sixth aspect, the present invention provides a fermented fertilizer obtained according to the above method.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] 1. This invention mixes three strains in a special ratio to prepare a compound fermentation agent that can efficiently ferment livestock and poultry manure, shortening the preparation time of human, livestock and poultry manure organic fertilizer and improving the fertilizer efficiency of organic fertilizer; at the same time, the addition of this compound agent can increase the activity of nitrogen conversion-related enzymes in compost, converting ammonia nitrogen and ammonium nitrogen in compost to a greater extent into nitrate nitrogen, and reducing the loss of nitrogen in the form of ammonia gas.

[0022] 2. Compared with traditional aerobic composting technology, this invention has better odor removal and nitrogen retention effects, and has the advantages of low cost, good effect, strong operability, no secondary pollution, environmental beautification, and promotion of the resource utilization of livestock and poultry manure. It can be widely promoted and applied.

[0023] 3. The compound microbial agent provided by this invention can promote the carbon and nitrogen conversion during the composting process of human, animal and poultry manure and agricultural straw, while increasing the composting temperature and compost maturity, thereby improving the quality of organic fertilizer.

[0024] Information on the preservation of biological materials:

[0025] Bacillus amyloliquefaciens, taxonomically named Bacillus amyloliquefaciens, was deposited on July 17, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.27935. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0026] Ochrobactrum pseudodogrignonense, taxonomic name: Ochrobactrum pseudodogrignonense; Latin name: Ochrobactrum pseudodogrignonense; This strain was deposited on July 17, 2023 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.27932, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0027] Penicillium chrysogenum, taxonomic name: Penicillium chrysogenum; Latin name: Penicillium chrysogenum; This strain was deposited on July 17, 2023 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.27934, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0028] Figure 1 It describes the morphology of Bacillus amyloliquefaciens;

[0029] Figure 2 It is a phylogenetic tree of Bacillus amyloliquefaciens;

[0030] Figure 3 It is a phylogenetic tree of *Pseudomonas aeruginosa*;

[0031] Figure 4 This is a morphological diagram of Penicillium chrysogenum C;

[0032] Figure 5 It is the phylogenetic tree of Penicillium chrysogenum C;

[0033] Figure 6 The changes in temperature (a) and Kjeldahl total nitrogen (b) in sheep manure compost over 50 days under different mixing ratios;

[0034] Figure 7 The changes in NH3 (a) and seed germination index (b) in sheep manure compost over 50 days under different mixing ratios;

[0035] Figure 8 The changes in nitratease (a) and urease (b) in sheep manure compost over 50 days under different mixing ratios;

[0036] Figure 9 The changes in protease in sheep manure compost over 50 days under different mixing ratios;

[0037] Figure 10 The changes in temperature (a) and Kjeldahl total nitrogen (b) before and after 42 days of cow manure composting under different mixing ratios are shown.

[0038] Figure 11 The changes in NH3 (a) and NH3 accumulation (b) before and after 42 days of cow manure composting under different mixing ratios are shown.

[0039] Figure 12 The changes in ammonium nitrogen (a) and N2O (b) before and after 42 days of cow manure composting under different mixing ratios are shown.

[0040] Figure 13 The changes in N2O accumulation (a) and nitrate nitrogen (b) before and after 42 days of cow manure composting under different mixing ratios are shown.

[0041] Figure 14 The changes in seed germination index before and after 42 days of cow manure composting under different mixing ratios are shown. Detailed Implementation

[0042] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0044] For the specific preparation method of the self-made decomposed sheep manure compost sample used in the embodiments of the present invention, please refer to the relevant article "Bioresource Technology, 335(2021)125282" published by the team of the present invention in 2021.

[0045] The record is found at https: / / doi.org / 10.1016 / j.biortech.2021.125282.

[0046] Example 1

[0047] Screening and isolation of nitrogen-fixing bacteria

[0048] 1. Isolation and initial screening of nitrogen-fixing bacteria

[0049] Take 10g of self-made, well-rotted sheep manure compost sample from Northwest A&F University. Place the 10g compost sample into a 250mL sterile Erlenmeyer flask containing 100mL of sterile water. After standing for 20min, shake thoroughly in a shaking incubator at 200rpm for 30min to prepare a bacterial suspension stock solution. After dilution, obtain 10g of the stock solution. -4 10 -5 and 10 -6 Sample solutions of different dilutions were prepared. These solutions were then spread onto sterilized Ashby nitrogen-free agar plates and incubated upside down at 37°C for 7 days. Finally, the strains were isolated and purified using the streak plate method. The obtained primary screening strains were inoculated onto LB slant agar and stored at 4°C.

[0050] 2. Secondary screening of nitrogen-fixing bacteria

[0051] The initially screened strains were inoculated into Ashby nitrogen-free liquid medium and cultured with shaking in a constant temperature incubator. Using uninoculated liquid medium as a control, the total nitrogen content in the fermentation broth of different strains was determined by the Kjeldahl method. The highly efficient nitrogen-fixing strains were stored in a -86°C refrigerator with 20% glycerol.

[0052] 3. Determination of bacterial antagonism

[0053] After activation, the strains were streaked in pairs on LB agar plates and cultured for 2–3 days. The growth at the crossover points was observed to determine whether there was any antagonistic effect between the strains. If there was an antagonistic reaction, they should not be mixed together to make compound microbial fertilizer. If there was no antagonistic reaction, the strains could be mixed and cultured.

[0054] The LB medium formula is as follows: NaCl 10g, yeast extract 5g, peptone 10g, distilled water 1000mL, pH 7.0, agar 20g (not added for liquid medium), sterilized at 121℃ for 20min.

[0055] Ashby nitrogen-free medium formula: KH2PO4 0.2 g, NaCl 0.2 g, MgSO4·7H2O 0.2 g, CaCO3 5.0 g, CaSO4·2H2O 0.1 g, mannitol 10 g, distilled water 1000 mL, pH 7.0, agar 20 g (omitted for liquid medium), sterilize at 121℃ for 20 min.

[0056] 4. Results and Analysis

[0057] Colonies with rapid growth, large size, and regular shape were selected from the initial screening medium. These colonies were repeatedly streaked to isolate and purify them into pure strains, including 6 fungi and 10 bacteria. After antagonistic reaction testing, the three strains with the strongest nitrogen-fixing ability and no mutual antagonism were selected and named Bacteria A, Bacteria B, and Fungus C, respectively. Some physiological and biochemical indicators of Bacteria A were determined according to the Bergers Manual of Bacterial Identification, and the results are shown in Table 1.

[0058] Table 1. Partial physiological and biochemical properties of bacteria A

[0059]

[0060]

[0061] Morphological analysis revealed that bacteria A belongs to the phylum Firmicutes, class Bacilli, family Bacillaceae, and genus Bacillus. It is Gram-positive, rod-shaped, and can form endospores. The endospores are oval with blunt ends, the sporangia are not enlarged, and they are central to subterminal in origin. They are motile, forming pale yellow, opaque colonies with a rough, raised surface and irregular edges. Figure 1 .

[0062] Phylogenetic comparison of bacteria A using molecular identification yielded the following results: Figure 2 As shown.

[0063] The results of some physiological and biochemical index determinations of bacteria B, based on the Bergers Manual of Bacterial Identification, are shown in Table 2.

[0064] Table 2. Partial physiological and biochemical properties of bacteria B

[0065]

[0066] Morphological analysis revealed that bacteria B belong to the phylum Proteobacteria, order Rhizobium, family Brucellosis, and genus Ochrobactrum. It is Gram-negative, rod-shaped, motile by peritrichous flagella, obligately aerobic, and strictly adhering to respiratory metabolism.

[0067] Phylogenetic comparison of bacteria B using molecular identification yielded the following results: Figure 3 As shown.

[0068] The results of some physiological and biochemical index determinations of fungus C are shown in Table 3.

[0069] Table 3. Partial physiological and biochemical properties of fungus C

[0070]

[0071] Morphological analysis revealed that fungus C belongs to the subphylum Deuteromycotina, class Hyphomycetes, order Hyphomycetes, and genus *Penicillium Chrysogenum*, and is a typical penicillin-producing fungus. The colony grows rapidly, with a blue-green colony structure and a yellowish-brown reverse side. Figure 4 .

[0072] Phylogenetic comparison of fungal C using molecular identification yielded the following results: Figure 5 As shown.

[0073] Example 2

[0074] Preparation of compound microbial agents

[0075] Specifically, the following steps are included:

[0076] S1, Single-strain fermentation:

[0077] Penicillium chrysogenum was inoculated onto PDA solid medium and cultured at 30°C for 72 h. A spore suspension was prepared with sterile water and counted using a hemocytometer. The concentration of the bacterial suspension was adjusted to 10. 8 cells / mL;

[0078] Bacillus amyloliquefaciens and Bacillus cereus were inoculated into LB liquid medium, incubated at 30°C for 48 h, and the bacterial cells were collected by centrifugation. The culture medium was then used to prepare 10 μL of culture medium with sterile water. 8 CFU / mL bacterial suspension.

[0079] S2. Preparation of compound microbial agent:

[0080] The three bacterial solutions of Bacillus amyloliquefaciens, Bacillus licheniformis, and Penicillium chrysogenum obtained in S1 are mixed in a volume ratio of 1:1:1 to obtain a compound bacterial agent. It should be noted that the three bacterial solutions of Bacillus amyloliquefaciens, Bacillus licheniformis, and Penicillium chrysogenum obtained in S1 can be mixed in a mass ratio of 1 to 3:1 to 3:1 to 3. The compound bacterial agent with the expected effect of the present invention can be obtained within this range. Those skilled in the art can choose the appropriate method according to their needs in actual operation.

[0081] S3. Adding a microbial community promoter: Add an equal mass of microbial community promoter to the compound microbial agent obtained in S2 to prepare the compound fermentation agent. The microbial community promoter specifically consists of: 15g wheat bran (as a carbon source), 5g soybean meal (as a nitrogen source), and 20mL Mandels's nutrient solution. Mix the three ingredients thoroughly, soak for 4 hours, and then sterilize at 115℃ for 30 minutes. Cool to room temperature before use. It should be noted that the mass ratio of wheat bran to soybean meal can vary from 2 to 4:1 to 2. The Mandels's nutrient solution only needs to submerge the mixture of wheat bran and soybean meal. Furthermore, the mass ratio of the compound microbial agent to the mixture of wheat bran and soybean meal can also vary from 1 to 4:1. Those skilled in the art can choose the appropriate ratio based on actual operation.

[0082] The required Mandels's nutrient solution formula is as follows: (NH4)2SO4 1.4 g / L, KH2PO4 2.0 g / L, CaCl2·2H2O 0.4 g / L, MgSO4 0.3 g / L, Peptone 1.0 g / L, NaCl 0.3 g / L, FeSO4 0.005 g / L, MnSO4 0.0016 g / L, ZnSO4·7H2O 0.0014 g / L, CoCl2 0.002 g / L.

[0083] Example 3

[0084] The compound microbial agent prepared in Example 2 was used for composting fermentation. First, a mixture of dried sheep manure and fruit tree branches (mainly a mixture of wolfberry, kiwi, and apple branches) collected from the Agricultural Experiment Station of Northwest A&F University was pulverized to a particle size of less than 5 mm, spread evenly on plastic sheeting, and moistened to a moisture content of 55%–60%. Based on the dry weight of the compost material, 2.5% of the compound microbial agent in different mixing ratios was added, stirred evenly, and then placed into a 60L aerobic fermentation tank to begin a 50-day aerobic composting experiment. During this period, the released NH3 was absorbed using an Erlenmeyer flask containing boric acid, and the daily NH3 release was measured. Simultaneously, the temperature of the compost at different depths was automatically recorded at 8:00 AM and 8:00 PM each day, and the average value was recorded as the daily compost temperature. For the first 20 days after the start of the experiment, the material was automatically aerated for 5 minutes every hour at a flow rate of approximately 400 L / (h·t dry weight). Thereafter, the aeration rate was increased to approximately 800 L / (h·t dry weight) until the end of the experiment. On the first day of the experiment, and on days 10, 20, 30, 40, and 50, the material was removed, spread on plastic sheeting, and manually turned over. Samples were then collected for the determination of relevant indicators.

[0085] Depend on Figure 6 As shown in Figure a, all treatments rapidly raised the temperature to above 50℃, indicating that the aerobic fermentation process of composting proceeded smoothly. However, the treatment with added microbial agents rapidly raised the temperature to above 50℃ on the 2nd to 3rd day after the start of composting, while the control treatment only reached a temperature close to 50℃ on the 5th day. Furthermore, the control treatment maintained a compost temperature above 55℃ for 6 days, while the other treatments with added microbial agents maintained a high-temperature period (above 55℃) for 10–14 days. The highest temperature during the high-temperature period in the control treatment was 58.4℃ (occurring on the 7th day), while the highest temperatures during the high-temperature period in the other treatments with added microbial agents ranged from 62.8 to 66.9℃ (occurring on the 4th to 7th days). All treatments in this experiment met the standards for the harmless treatment of livestock and poultry manure, but the treatment with added microbial agents was more conducive to the rapid heating and complete high-temperature harmless treatment of the compost material.

[0086] Depend on Figure 6As shown in b, the control treatment exhibited a typical Kjeldahl total nitrogen (TNI) change pattern during composting, namely, a significant decrease in TNI during the high-temperature composting period (due to NH3 volatilization loss; a decrease in TNI was observed on day 10 of this experiment), followed by a gradual increase in TNI as the composting process progressed. In contrast, the TNI content in the treatments with added microbial agents showed a generally continuous increase throughout the experiment. At the end of the 50-day composting period, the TNI content in the control treatment was 18.05 g / kg, while the TNI content in the other treatments with added microbial agents ranged from 19.94 to 22.65 g / kg. The addition of microbial agents increased the TNI content of the compost product by 10.47% to 25.48%. This suggests that adding compound microbial agents to the compost material is beneficial for increasing the nitrogen content of the product compared to the control treatment, a point also confirmed by the changes in NH3 during the experiment. Throughout the experiment, NH3 release in each treatment was mainly concentrated in the first two weeks of composting, which is consistent with the typical pattern of NH3 release during aerobic fermentation of organic waste; among them, the control treatment showed a significantly higher NH3 release during this stage than the treatments with added microbial agents. Figure 7 a). This result confirms that adding compound microbial agents can effectively reduce the release and volatilization of NH3 during composting, thereby promoting the nitrogen retention capacity of compost products.

[0087] To further verify the effect of adding microbial agents on the composting process, the germination index of *Hedyotis diffusa* seeds in the relevant samples was measured, and the results are as follows: Figure 7 As shown in b, during the composting process, the seed germination index of each treatment was low in the early stage of composting. This is related to the production of NH3 and small-molecule organic acids, which have certain biotoxicity, at this stage. This change is consistent with the typical change pattern of maturity in the aerobic fermentation process of organic waste. As the composting process is prolonged, the amount of NH3 produced decreases and the small-molecule organic acids are decomposed and mineralized, and the seed germination index of the compost samples gradually increases. Its toxicity to plant seed germination weakens, and the compost gradually tends to mature. According to the People's Republic of China Agricultural Industry Standard "Organic Fertilizer" (NY525-2021), the seed germination index of the product should be ≥0.7. The control treatment reached maturity at 0.74 after 50 days; the treatment with added microbial agents reached maturity at 30 days (at which time the seed germination index was between 0.72 and 0.81), and the seed germination index was even higher at 0.89 to 1.09 on the 50th day of composting. This result also shows that adding compound microbial agents can promote the maturation of compost products and shorten the maturation time, which can be reduced from 50 days to 30 days in this experiment.

[0088] Composting, as a bio-mediated biochemical reaction process, relies heavily on enzymes secreted by microorganisms for nutrient cycling. To further illustrate the promoting effect of adding compound microbial agents on the composting process, the main enzymes involved in nitrogen transformation were also measured in the experiment. Nitrate reductase, an oxidoreductase, is a type of NH4+ that promotes nitrogen transformation.+ Nitrogen (N-N) is converted into stable nitrogenous substances and stored in aerobic fermentation products. Therefore, nitrate reductase can reflect the intensity of nitrogen conversion during aerobic fermentation. Nitrate nitrogen in the natural environment induces changes in nitrate reductase activity, which often shows a positive correlation with the nitrogen concentration in the medium. For example... Figure 8 As shown in Figure a, in this experiment, the nitrate enzyme content was relatively low in the early stage of aerobic fermentation, and then gradually increased with the extension of fermentation time. At the end of 50 days of fermentation, the nitrate enzyme content in the treatment with added compound microbial agent was between 1.08 and 1.37 g / kg / d, which was much higher than the 0.72 g / kg / d in the control treatment, indicating that the added microbial agent had a positive effect on maintaining nitrate enzyme activity during fermentation. In the aerobic fermentation of livestock and poultry manure, urease can catalyze the conversion of amide compounds into ammonia, which can reflect the mineralization process of organic nitrogen and plays a crucial role in nitrogen cycling. Figure 8 As shown in b, the urease activity in all treatments reached its peak around day 20, indicating that microorganisms rapidly converted organic nitrogen during this stage, which was also reflected in the trend of total nitrogen changes. Subsequently, urease activity decreased with increasing fermentation time until the end of fermentation. At the end of 50 days of aerobic fermentation, the urease content in the treatment with added compound microbial agents ranged from 11.49 to 16.94 g / kg / h, significantly higher than the 8.61 g / kg / h in the control treatment, indicating that the treatment with added compound microbial agents had relatively abundant nitrogen and higher related microbial activity. Proteases are a general term for enzymes secreted by microorganisms that hydrolyze protein peptide chains; they are closely related to the decomposition of nitrogenous substances in compost materials and the transformation of amino acids and other nitrogenous components. Figure 9 As shown, the protease activity in all treatments gradually decreased with increasing fermentation time until the end of the fermentation process. Protease activity was higher in the early stages of aerobic fermentation, which is related to the rapid degradation of proteins and peptides to ammonia during this period. Subsequently, with increasing fermentation time, the protease activity in each treatment gradually decreased until the end of the fermentation process, mainly due to the gradual mineralization and reduction of nitrogen-containing substances such as proteins, peptides, and amino acids. At the end of 50 days of aerobic fermentation, the protease content in the treatment with added compound microbial agents ranged from 28.86 to 39.44 mg / g / d, which was higher than the 27.49 mg / g / d in the control treatment. Based on these experimental results, it can be seen that compared with the control, the contents of nitratease, urease, and protease in the other treatment groups with added compound microbial agents were all higher than those in the control treatment without added compound microbial agents. This indicates that adding compound microbial agents can increase the activity of enzymes involved in nitrogen transformation, and it plays an important regulatory role in the nitrogen transformation of compost materials.

[0089] Example 4

[0090] The compound microbial agent from Example 2 was used for composting in a pilot-scale aerobic fermenter. First, wheat straw collected from the First Agricultural Experiment Station of Northwest A&F University was pulverized (<2mm fragments). Then, it was thoroughly mechanically mixed with fresh dairy cow manure (high moisture content, approximately 90%) collected from the Third Agricultural Experiment Station of Northwest A&F University until the particle size was <5mm, adjusting the moisture content to 55%–60% (achieved by adding wheat straw powder; the carbon-to-nitrogen ratio was not adjusted in this experiment). Subsequently, based on the dry weight of the compost material, 5% of the compound microbial agent in different mixing ratios was added to the compost material. After thorough mixing, the mixture was placed into 180L aerobic fermenters to begin a 42-day aerobic composting experiment. For ease of description, the treatment without added microbial agent was designated as the control, and the treatments with added compound microbial agent were designated as 1:1:1, 1:1:3, 1:3:1, 1:3:3, 3:1:1, and 3:3:1, for a total of seven treatments. During the experiment, the released NH3 was absorbed using Erlenmeyer flasks containing boric acid, and the daily NH3 release was measured. Simultaneously, the temperature of the pile at different depths was automatically recorded at 8:00 AM and 8:00 PM daily, and the average value was taken. For the first 20 days of the experiment, automatic aeration was carried out for 5 minutes every hour at a flow rate of approximately 400 L / (h·t dry weight), thereafter increasing the aeration rate to approximately 800 L / (h·t dry weight) until the end of the experiment. On days 1, 3, 7, 14, 21, 28, 35, and 42 of the experiment, the material was removed, spread on plastic sheeting, and manually turned over, while samples were collected for analysis.

[0091] Depend on Figure 10 As shown in Figure a, all treatments rapidly raised the temperature to above 50℃, indicating that the aerobic fermentation process of composting proceeded smoothly. The control treatment reached a high-temperature period of above 55℃ on the 5th day after composting began, while the treatments with added microbial agents rapidly raised the temperature to above 55℃ on the 2nd to 3rd day. Furthermore, the control treatment maintained a compost temperature above 55℃ for 7 days, while the other treatments with added microbial agents maintained it for 10–18 days. The highest temperature during the high-temperature period in the control treatment was 56.6℃ (occurring on the 9th day), while the highest temperatures during the high-temperature period in the other treatments with added microbial agents ranged from 61.4 to 70.2℃ (occurring on the 4th to 9th days). All treatments in this experiment met the requirements of the harmless treatment standard for livestock and poultry manure (GB / T 36195-2018), but the treatment with added compound microbial agents was more conducive to the rapid heating and complete high-temperature harmless treatment of the compost material. In addition, from... Figure 10As shown in b, all control treatments exhibited a typical pattern of total nitrogen variation in compost: a decrease in total nitrogen due to NH3 volatilization loss during the high-temperature composting period, followed by a gradual increase in total nitrogen as the composting process progressed. Compared to the control treatment, which maintained a consistently low total nitrogen level (11.64–12.39 g / kg) from day 3 to day 14, the Kjeldahl total nitrogen content in the inoculant-added treatments showed a relatively high level after day 3. By day 50, at the end of composting, the total nitrogen content in the control treatment was 18.73 g / kg, while the total nitrogen content in the other inoculant-added treatments ranged from 21.13 to 22.78 g / kg. The inoculant-added treatments increased the total nitrogen content of the compost product by 12.81%–21.62%. This suggests that adding a compound inoculant to the compost material is beneficial for increasing the nitrogen content of the product compared to the control treatment, a point also confirmed by the changes in NH3 during the experiment. Throughout the experiment, NH3 release from each treatment was mainly concentrated in the first two weeks of composting, which is consistent with the typical pattern of NH3 release during aerobic fermentation of organic waste; among them, the control treatment showed significantly higher NH3 release during this stage than the treatment with added microbial agents. Figure 11 a) and the cumulative NH3 release in the control treatment during the entire composting process was 11.52 g, while the cumulative NH3 release in the treatment with added compound microbial agent was between 2.78 and 4.57 g. Figure 11 (b) The cumulative release of NH3 during the composting process was reduced by 60.33%–75.87%. This result also confirms that adding compound microbial agents can effectively reduce the release and volatilization of NH3 during composting, thereby promoting the nitrogen retention capacity of compost products. To further analyze the reasons, the experiment further analyzed ammonium nitrogen (NH4+). + The changes in ammonium nitrogen (Am-N) content were analyzed, and it was found that the ammonium nitrogen content showed a trend of first rapidly increasing and then gradually decreasing throughout the composting process. Figure 12 a) Before day 21, the treatment with the added compound microbial agent had a higher ammonium nitrogen content than the control treatment. After day 21, the ammonium nitrogen content in the treatment with the added compound microbial agent decreased rapidly, reaching 0.14–0.30 g / kg by day 28, below the limit for ammonium nitrogen content in mature compost (<0.4 g / kg). In contrast, the ammonium nitrogen content in the control treatment was 0.66 g / kg on day 28, 0.49 g / kg on day 35, and decreased to 0.38 g / kg by day 42. This result suggests that adding a compound microbial agent to compost materials can promote the conversion of NH3 to NH4 during the high-temperature period. + -N fixation reduces volatilization loss and effectively promotes NH4+ filtration during the composting cooling and maturation periods. + -N undergoes further transformation through nitration. Therefore, the N2O release, N2O accumulation, and nitrate nitrogen (NO3) in each treatment were further measured and analyzed. --N) variation pattern. For example Figure 12 As shown in b, during the composting process, the N2O released by each treatment in the early stage of composting was very little (almost undetectable), and gradually increased after the pile temperature dropped below 50℃, which is related to the inability of nitrifying bacteria to withstand high temperatures. Subsequently, as the pile temperature decreased and the composting time increased, the N2O release gradually and rapidly increased, and then rapidly decreased during the composting maturation period. Throughout the entire composting process, the cumulative N2O release in the control treatment was 1.32g, while the cumulative N2O release in the treatment with the added compound microbial agent ranged from 0.79 to 1.08g. Figure 13 a) Treatment with compound microbial agents can reduce N2O accumulation by 24.0%–40.2%. Meanwhile, as the composting process progresses, the nitrate nitrogen content in all treatments remains relatively low during the high-temperature period, but rapidly increases during the cooling and maturation periods. Figure 13 (b) By the end of composting, the nitrate nitrogen content in the control treatment was 0.82 g / kg, while the cumulative N2O content in the treatment with added compound microbial agents was between 1.16 and 1.40 g / kg. These results also confirm that adding compound microbial agents is beneficial to the aerobic composting process and the conversion and fixation of nitrate nitrogen.

[0092] If according to NH4 + -N / NO3 - The ratio of NH4+ to nitrogen (<0.5 indicates complete decomposition, 0.5-3.0 indicates basic decomposition, and >3.0 indicates immaturity) was used to determine compost maturity. On day 28, the ratio for the control treatment was 1.32, indicating basic decomposition, while the ratio for the treatment with added compound microbial agents was 0.18-0.30, indicating complete decomposition. On day 35, the ratio for the control treatment was 0.70, indicating basic decomposition, while the ratio for the treatment with added compound microbial agents was 0.12-0.14, indicating complete decomposition. Finally, on day 42, the ratio for the control treatment was 0.46, indicating complete decomposition, while the ratio for the treatment with added compound microbial agents was 0.08-0.10, also indicating complete decomposition. This demonstrates that adding compound microbial agents promotes compost maturity. If calculated according to NH4+... + -N / NO3 - The ratio of -N is used to judge the maturity of the product. Adding a compound microbial agent can shorten the composting cycle from 42 days to 28 days. To further confirm this conclusion, the germination index of mustard seeds in the relevant samples was further measured in the experiment. Figure 14During the composting process, the seed germination index of each treatment was low in the early stage of composting. This is related to the production of NH3 and small-molecule organic acids, which have certain biotoxicity, at this stage. This change is consistent with the typical change pattern of maturity in the aerobic fermentation process of organic waste. As the composting process is prolonged, the amount of NH3 produced decreases and the small-molecule organic acids are decomposed and mineralized, and the seed germination index of the compost samples gradually increases. Its toxicity to plant seed germination weakens, and the compost gradually tends to be mature. According to the People's Republic of China Agricultural Industry Standard "Organic Fertilizer" (NY525-2021), the seed germination index of mature products should be ≥0.70. The treatment with added microbial agent was close to maturity on day 21 (at which time the seed germination index was between 0.70 and 0.81), and the seed germination index reached 0.82 to 0.90 on day 28 of composting, and 0.90 to 1.06 on day 42. In contrast, the control treatment only met the maturity requirement on day 42, with a seed germination index of only 0.73. This result also indicates that adding compound microbial agents can promote the decomposition of compost products and shorten the decomposition time. If the decomposition status of compost products is judged according to the seed germination index ≥0.7 specified in "Organic Fertilizer" (NY525-2021), adding compound microbial agents can shorten the composting cycle from 42 days to 28 or even 21 days.

[0093] Example 5

[0094] Since Example 3 was conducted in a laboratory-scale automated aerobic fermenter and Example 4 in a pilot-scale aerobic fermenter, both yielding essentially the same application results, Example 5 employed an engineering-grade semi-open fermentation tank composting process. Specifically, at a livestock and poultry manure treatment plant in Yangling, Shaanxi Province, 3 tons of wet livestock and poultry manure (a mixture of pig, sheep, and chicken manure), 0.5 tons of dry toilet human excrement (wet manure), and 9 tons of mixed dry powder materials (mainly sawdust, rice husks, and corn stalk bran) provided by the plant were thoroughly mixed using a mixer. The moisture content was adjusted to approximately 55%, the carbon-to-nitrogen ratio to approximately 1:35, and the particle size of the mixture was ensured to be less than 1 cm. The mixture was then loaded into a semi-open fermentation tank and tightly covered with plastic sheeting for a 30-day aerobic composting experiment. Similarly, a control treatment and treatments with different mixing ratios of microbial agents were set up, totaling 7 treatments. One of them served as the control group (no microbial agent added), while the others were treated with different ratios of compound microbial agents. For ease of description, these were recorded as 1:1:1, 1:1:3, 1:3:1, 1:3:3, 3:1:1, and 3:3:1 treatments, totaling 6 compound microbial agent treatments. During the experiment, the temperature at the surface 30 cm and center 60 cm of the pile was measured daily using a Beckman precision thermometer, and the average value was recorded. The pile temperature change was recorded. Simultaneously, the pile was mechanically turned once a day, and after each turning, it was tightly covered with plastic sheeting. After 30 days of primary fermentation composting, relevant samples were collected, and indicators such as pH, electrical conductivity (EC) (solid-liquid ratio 1:5), total nitrogen, total phosphorus, total potassium, pH, organic matter, and the germination index of *Syzygium aromaticum* seeds were measured. The results are shown in Table 4.

[0095] Table 4. Determination of relevant indicators in compost products of each treatment group after 30 days.

[0096] Comparison 18.76 11.13 6.30 8.83 1.76 0.58 1:1:1 23.12 13.74 9.13 8.27 1.85 0.96 1:1:3 22.54 13.69 9.17 8.43 2.17 0.91 1:3:1 22.81 13.03 8.81 8.40 1.86 0.86 1:3:3 22.73 12.93 8.89 8.45 1.89 0.97 3:1:1 23.06 13.58 9.03 8.38 2.04 0.98 3:3:1 22.86 12.95 8.67 8.51 1.99 1.03

[0097] Table 4 shows that after 30 days of composting, the control treatment had relatively low total nitrogen, phosphorus, and potassium content, high pH, ​​and low seed germination index (not reaching the maturity standard), while the treatment with added compound microbial agent had relatively high total nitrogen, phosphorus, and potassium content, low pH, and high seed germination index (all reaching the maturity standard), both meeting the national standard for organic fertilizers (NY525-2021). Therefore, on an engineering scale, the method provided by this invention can effectively promote nitrogen retention and nutrient fixation in composting and achieve the maturity of compost products.

[0098] The above results show that the method provided by the present invention can effectively reduce the emissions of ammonia and nitrous oxide in composting, has good nitrogen retention and nutrient fixation effects in composting, can improve the quality of compost products, beautify the environment, reduce pollution, and promote the resource utilization of organic waste such as human and animal manure and agricultural straw.

[0099] It should be noted that the steps and methods used in the claims of this invention are the same as those in the above embodiments. To avoid redundancy, preferred embodiments are described in this invention. However, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0100] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A compound microbial agent for reducing nitrogen loss in composting, characterized in that, It is composed of Bacillus amyloliquefaciens CC-LRH6 ( Bacillus amyloliquefaciens ), Pseudomonas aeruginosa CN-LRH3 ( Ochrobactrum pseudogrignonense ) and Penicillium chrysogenum CN-LRH5 ( Penicillium chrysogenum The compound is composed of a mixture of *Bacillus amyloliquefaciens* CC-LRH6, *Bacillus cereus* CN-LRH3, and *Penicillium chrysogenum* CN-LRH5, all of which are deposited at the China General Microbiological Culture Collection Center (CGMCC). The accession number for *Bacillus amyloliquefaciens* CC-LRH6 is CGMCC NO.27935, for *Bacillus cereus* CN-LRH3 it is CGMCC NO.27932, and for *Penicillium chrysogenum* CN-LRH5 it is CGMCC NO.27934. The volume ratio of the seed culture of *Bacillus amyloliquefaciens* CC-LRH6, the seed culture of *Bacillus cereus* CN-LRH3, and the bacterial culture of *Penicillium chrysogenum* CN-LRH5 in the compound microbial agent is 1~3:1~3:1~3, and the effective viable count of both the seed culture of *Bacillus amyloliquefaciens* CC-LRH6 and the seed culture of *Bacillus cereus* CN-LRH3 is 10. 8 The concentration of the Penicillium chrysogenum CN-LRH5 bacterial culture was 10 CFU / mL. 8 per mL.

2. A method for preparing the compound microbial agent according to claim 1, characterized in that, Includes the following steps: The Bacillus amyloliquefaciens CC-LRH6 and the Bacillus cereus CN-LRH3 were respectively inoculated into LB liquid medium and cultured to obtain seed culture of both. The Penicillium chrysogenum CN-LRH5 was inoculated into PDA solid medium and cultured. The resulting bacterial solution was mixed with the seed solution of Bacillus amyloliquefaciens CC-LRH6 and the seed solution of Bacillus cereus CN-LRH3 to obtain the compound bacterial agent.

3. The preparation method according to claim 2, characterized in that, The seed culture was obtained by culturing at 25-30℃ and 120-180 rpm for 24-48 hours.

4. The preparation method according to claim 2, characterized in that, The effective viable bacteria count of both seed liquids was 10. 8 CFU / mL, the concentration of the bacterial culture is 10. 8 The volume ratio of the seed culture of Bacillus amyloliquefaciens CC-LRH6, the seed culture of Bacillus cereus CN-LRH3, and the bacterial culture of Penicillium chrysogenum CN-LRH5 is 1~3∶1~3∶1~3.

5. The use of the compound microbial agent according to claim 1 in composting fermentation.

6. The use according to claim 5, characterized in that, The compound microbial agent is used to reduce nitrogen loss during composting fermentation.

7. A microbial inoculant, characterized in that, It includes the compound microbial agent as described in claim 1, and microbiologically acceptable excipients.

8. The microbial agent according to claim 7, characterized in that, The auxiliary material is a microbial community promoter, which is wheat bran, soybean meal, and Mandels's nutrient solution. The mass ratio of wheat bran to soybean meal is 2-4:1-2. The Mandels's nutrient solution submerges the mixture of wheat bran and soybean meal. The mass ratio of the compound microbial agent to the mixture of wheat bran and soybean meal is 1-4:

1.

9. A method for composting fermentation using the compound microbial agent according to claim 1, characterized in that, The compound microbial agent is added to the compost material, and aerobic fermentation is carried out for 30 to 50 days. The amount of compound microbial agent added is 2.5% to 5% based on the dry weight of the compost material. The moisture content of the compost material is adjusted to 55% to 60% before adding the compound microbial agent.

10. A fermented fertilizer obtained by the method according to claim 9.

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