Thermophilic ammoniation compound microbial agent, fertilizer and application

Abstract

The invention belongs to the field of resource utilization of livestock and poultry manure, and particularly relates to a thermophilic ammoniation compound microbial agent, a fertilizer and application. The thermophilic ammoniation compound microbial agent is prepared by compounding lysinibacillus spp., bacillus megatherium and white-rot fungi, the preservation number of the lysinibacillus sphaeroides is CCTCC NO: M 20242776, the preservation number of the bacillus megaterium is CCTCC NO: M 20242777, the preservation number of the white-rot fungi is CCTCC NO: M 20242778, and the three strains are all preserved in the China Center for Type Culture Collection. Tests prove that the thermophilic ammoniation compound microbial agent shows higher ammoniation activity at 60 DEG C, and can be used for promoting the compost recycling process and improving the compost product quality.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of resource utilization of livestock and poultry manure, and particularly relates to a thermophilic ammonification composite microbial agent, a fertilizer and a use. BACKGROUND

[0002] The annual output of livestock and poultry manure is as high as more than 3 billion tons, which has the dual nature of pollution and resource. Resource utilization of livestock and poultry manure is an effective means to cope with the problems of pollution and waste of resources. With the rapid development of intensive farming, the output of livestock and poultry manure has increased dramatically, causing serious pollution to the environment and leading to the spread of pathogenic bacteria. Therefore, converting livestock and poultry manure into valuable resources not only can improve the breeding environment and pollution of the ecological environment, but also can improve the utilization efficiency of livestock and poultry manure resources, so that livestock and poultry manure becomes a high-value product. Composting technology is one of the important ways to utilize livestock and poultry manure resources and harmless. It can convert easily degradable organic matter into high-value compost products and organic fertilizer through a series of spontaneous biochemical reactions. In addition, the benefits of composting to the environment, economy and agriculture are recognized. As a biological treatment technology that can resource livestock and poultry manure, composting has the advantages of short cycle, simple process flow, small land occupation, low investment cost and mature technology, and has broad development prospects and market demand, and is widely used in resource utilization and harmless treatment of livestock and poultry manure. In addition, composting can convert livestock and poultry manure into stable, sanitary and high-nutrient organic fertilizer through a series of biochemical reactions. In the composting process, organic matter is converted into humus (humus, etc.) through the action of microorganisms. The high-value humus formed by livestock and poultry manure can be used as a soil conditioner to improve soil fertility and promote plant growth, and can become a valuable agricultural fertilizer commodity through development.

[0003] Microorganisms play an irreplaceable role in the mineralization and decomposition of compost and the composting process, and microorganisms with specific functions can effectively promote the decomposition and synthesis of specific substances in the composting process. According to previous research reports, ammonification microorganisms are mainly involved in the transformation and mineralization of nitrogen, which can convert macromolecular nitrogen-containing substances into low-molecular-weight nitrogen-containing substances through depolymerization, such as Lysinibacillus fusiformis Fungi can cross the microsites of soil and secrete extracellular enzymes to depolymerize nitrogen-containing compounds, participating in the depolymerization of macromolecular nitrogen-containing substances. In the related reports of composting, bacteria ( Bacillus and Pseudomonas ) and fungi ( Penicillium and Acremonium ) have been proved to have high ammonification activity.

[0004] Ammonification is a key step in the mineralization of organic nitrogen and a rate-limiting step between the conversion of organic nitrogen to inorganic nitrogen. Ammonification microorganisms and the volatilization of ammonia (NH3) and ammonium (NH4+ Ammonifying microorganisms are involved in the formation of nitrogen compounds and are a crucial step in nitrogen transformation. They determine the transfer equilibrium between nitrogen-related redox reactions. Ammonifying microorganisms play a key rate-limiting role among nitrogen-transforming microorganisms. They degrade nitrogen-containing compounds (urea, amino acids, nicotine, etc.) into small, absorbable organic nitrogen molecules, which are then bioavailable (decomposition and synthesis). Most microorganisms exhibit ammonifying activity, including bacteria and fungi. For example, Proteobacteria and Firmicutes contain members extremely important to the global carbon and nitrogen cycle, such as Pseudomonas and Bacillus, which are widely distributed and have high ammonifying activity. Furthermore, ammonifying microorganisms not only rapidly initiate the fermentation process, generating heat through their metabolic activities and increasing the compost temperature, thereby accelerating the reproduction and activity of other microorganisms, but their activity also promotes nitrogen transformation and fixation in compost, providing the material basis for the formation of high-value compost products.

[0005] However, the activity of most ammonifying microorganisms is often inhibited under the high-temperature environment of composting. Encouragingly, numerous studies have shown that inoculating thermophilic microorganisms can still exhibit high activity under the high-temperature conditions of composting. Furthermore, due to the vast diversity of microorganisms involved in composting, inoculating a single microbial species does not represent the contribution of that functional microbial population to the composting process. Numerous studies have indicated that while inoculating a composite microbial community alters a particular microbial community in the environment, other microbial communities may take its place, thereby enhancing the function of that specific microbial community. Currently, there is a lack of thermophilic ammonifying composite microbial agents that can effectively overcome the inhibitory effect of high temperatures on biotransformation during the thermophilic stage of composting. Therefore, this invention aims to provide a novel thermophilic ammonifying composite microbial agent to promote the resource utilization process of cow manure compost and improve the quality of compost products. Summary of the Invention

[0006] Based on the above technical problems, the present invention provides a thermophilic ammonification compound microbial agent, which has a good composting effect.

[0007] The specific technical solution provided by this invention is as follows: In a first aspect, the present invention provides a thermophilic ammonifying compound microbial agent, which is composed of *Bacillus fusiformis* (a type of lysine-containing bacterium). Lysinibacillus fusiformis ), Bacillus megaterium ( Bacillus megaterium It is a compound of Bacillus fusiformis and Phanerochaete chrysosporium; the accession number of Bacillus fusiformis is CCTCC NO: M 20242776, the accession number of Bacillus megaterium is CCTCC NO: M 20242777, and the accession number of Phanerochaete chrysosporium is CCTCC NO: M 20242778. All three strains are deposited at the China Center for Type Culture Collection.

[0008] In a preferred embodiment of the present invention, the volume ratio of *Bacillus spindleii* bacterial solution, *Bacillus megaterium* bacterial solution, and white-rot fungal solution is 1.1~1.2:1.4~1.6:0.9~1, and the effective viable count of the thermophilic ammonifying composite microbial agent is 1×10⁻⁶. 8 ~10 9 cfu / mL.

[0009] In a preferred embodiment of the present invention, the thermophilic ammonifying compound microbial agent is composed of Bacillus spindleii, Bacillus megaterium, white-rot fungi, and microbiologically acceptable excipients.

[0010] In a second aspect, the present invention provides a method for preparing a thermophilic ammonifying composite microbial agent, comprising the following steps: After activating Bacillus fusiformis, Bacillus megaterium, and white-rot fungi respectively, they were inoculated into Landy liquid medium and cultured together. After enrichment culture, the enriched culture solution was collected to obtain the thermophilic ammonification compound microbial agent.

[0011] In a third aspect, the present invention provides the application of the aforementioned thermophilic ammonification compound microbial agent in composting.

[0012] In a preferred embodiment of the present invention, the thermophilic ammonification compound microbial agent or the microbial agent is used to promote the ammonification process.

[0013] In a preferred embodiment of the present invention, the thermophilic ammonification compound microbial agent or the microbial agent is used to promote the composting process and improve the quality of compost products.

[0014] In a fourth aspect, the present invention provides a fertilizer obtained by inoculating the thermophilic ammonifying compound microbial agent into compost material for composting fermentation.

[0015] In a preferred embodiment of the present invention, the compost material is biomass waste or livestock excrement. For example, biomass waste may be straw, and livestock excrement may be cow dung.

[0016] In a fifth aspect, the present invention provides the use of the fertilizer described herein in promoting plant growth.

[0017] In a preferred embodiment of the present invention, the fertilizer is used to promote the accumulation of chlorophyll and biomass in plants, or to promote the growth of plant height and root length.

[0018] In a preferred embodiment of the present invention, the plant is Chinese cabbage.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention isolated and identified three highly efficient ammonifying microorganisms: thermophilic Z1 (Bacillus fusiformis), thermophilic Z2 (Bacillus megaterium), and thermophilic F1 (white-rot fungus). These three strains were mixed to prepare a thermophilic ammonifying composite microbial agent (Amm-4). Furthermore, the Amm-4 agent was further stabilized through high-temperature gradient acclimation (30℃~60℃). Under temperature gradient acclimation, the fungi and bacteria formed a stable biofilm structure in the Amm-4 agent, resulting in higher ammonification activity (NH4+) at high temperatures (60℃). + (204.83±6.73 mg / L). Experiments have demonstrated that the Amm-4 inoculant, when applied in composting, can promote the resource utilization process and improve the quality of compost products. Specifically, compared to the control group, inoculating cow manure with Amm-4 during composting reduced NH3 emissions by 23.3% and N2O emissions by 61.1%, increased total Kjeldahl nitrogen (TKN) content, promoted the degradation of total organic carbon (TOC) and dissolved organic carbon (DOC) during cow manure composting, reduced CO2 emissions by 32.4% and CH4 emissions by 18.0%, enhanced the activity of carbon conversion-related enzymes, and improved the humification degree of compost products. Furthermore, pot experiments were conducted to evaluate the quality of the compost products. The results showed that, compared to Con, the Amm-4-treated compost products significantly increased the chlorophyll, plant height, root length, and biomass of Chinese cabbage by 36.76%, 98.18%, 52.62%, and 84.04%, respectively.

[0020] Information on the preservation of biological materials: Spindle-shaped lysine-containing Bacillus, Latin name is Lysinibacillus fusiformis Its taxonomic name is: *Bacillus fusiformis*, and its preservation number is CCTCC NO: M 20242776.

[0021] The white-rot fungus, with the Latin name Phanerochaete chrysosporium and the taxonomic name Phanerochaete chrysosporium, has the accession number CCTCC NO: M 20242778.

[0022] Bacillus megaterium, Latin name Bacillus megaterium Its taxonomic name is Bacillus megaterium, and its preservation number is CCTCC NO: M 20242777.

[0023] All three strains were deposited at the China Center for Type Culture Collection on December 10, 2024, at Wuhan University, Wuhan, China. Attached Figure Description

[0024] Figure 1 These are the PCR identification results for Z1 (thermophilic bacteria), Z2 (thermophilic bacteria), and F1 (thermophilic fungi); Figure 2 It is the key ammonification gene—urease gene—of Z1 (thermophilic bacteria), Z2 (thermophilic bacteria), and F1 (thermophilic fungi). UreC ) identification; Figure 3 The phylogenetic tree based on the neighbor-joining method of gene sequence showed that strains Z1, Z2 and F1 were Bacillus spindleii, Bacillus megaterium and white-rot fungi, respectively; Figure 4 It is the result of antagonism between Z1, Z2, and F1; Figure 5 These are the Gram staining results of bacteria Z1 and Z2 and the morphological observation results of fungus F1; Figure 6 These are the growth curves of strains Z1, Z2, and F1; Figure 7 The results are the Nessler colorimetric reactions of four ammonifying agents (Amm-1, Amm-2, Amm-3, and Amm-4) at incubation temperatures of 30°C and 60°C. Figure 8 The ammonification performance (a) and growth (b) of four enriched ammonifying microbial agents at different temperatures are shown. Figure 9 These are scanning electron microscope (SEM) results of four ammoniating bacterial agents (Amm-1, Amm-2, Amm-3, and Amm-4) after being acclimated at 60°C. Figure 10 The results are EDS values ​​after the thermophilic ammonifying compound microbial agent (Amm-4) has been acclimatized at 60℃. Figure 11 The effects of inoculating thermophilic ammonifying compound microbial agent (Amm-4) on the composting process include: a) temperature, b) pH, c) electrical conductivity, and d) organic matter content. Figure 12 Amm-4 is inoculated into NH4 during the composting process. + -N(a), NO3 − The contents of -N(b), TKN(c) and NH4 + -N / NO3 − -N(d) change; Figure 13 The effects of Amm-4 inoculation during composting on the cumulative emissions of NH3 (a) and N2O (b); Figure 14 The effect of Amm-4 inoculation during composting on total organic carbon (a) and dissolved organic carbon (b); Figure 15The figure shows the effect of Amm-4 inoculation during composting on the cumulative emissions of CO2 and CH4; different letters in the figure indicate significant differences. Figure 16 The effects of Amm-4 inoculation during composting on polyphenol oxidase activity (a), amylase activity (b), laccase activity (c), and cellulase activity (d) were investigated. Figure 17 The effects of Amm-4 inoculation during composting on the quality of compost products (pot experiment) are shown. a, chlorophyll, b, plant height, c, root length, d, biomass; different values ​​of a1 and b1 in the figure indicate significant differences. Figure 18 The effect of Amm-4 inoculation on the degree of humification during composting is shown in the following figures: a) Day 1 of composting; b) Day 42 of composting in the control group; c) Day 42 of composting in the Amm-4 treatment group; d) Fluorescence region integral. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to specific embodiments, but the present invention is not limited thereto.

[0026] 1. Main reagents Bacterial culture medium (LB fortified): 3.0 g / L beef extract, 10.0 g / L peptone, 5.0 g / L NaCl, 10 g / L agar and 1.0 L sterile distilled water, pH 7.2~7.4.

[0027] Fungal culture medium (PDA): 5 g potato starch, 20 g glucose, 0.1 g chloramphenicol, 15 g / L agar powder and 1.0 L sterile distilled water.

[0028] Ammoniation enrichment medium (M1): 5 g / L peptone, 0.5 g / L KH2PO4, 0.2 g / L K2HPO4, 0.5 g / L MgSO4·7H2O, 15 g / L agar and 1.0 L sterile distilled water, pH 7.2.

[0029] Landy medium: 20.0 g glucose, 1.0 g yeast extract, 5.0 g L-glutamic acid, 2 mg L-phenylalanine, 1.0 g L-tryptophan, 0.5 g potassium chloride, 1.0 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 5 mg manganese sulfate tetrahydrate, 0.16 mg copper sulfate heptahydrate, 0.15 mg ferrous sulfate heptahydrate, and 1.0 L sterile distilled water.

[0030] 2. Method 2.1 Isolation and purification of strains Twenty-three bacterial strains and nine fungal strains were initially screened from cow manure samples collected by Ningxia Jingtian Livestock Farm and various cattle farms in Shaanxi Province using enrichment culture methods. Of these, 15 bacterial strains were derived from fresh cow manure (within 5 minutes of excretion, 25℃~30℃); 8 bacterial strains were derived from the thermophilic stage of cow manure composting (50℃~60℃); and 9 fungal strains were derived from the maturation stage of cow manure composting (30℃~40℃). Further screening using Nessler's reagent colorimetric reaction ultimately identified the two bacteria and one fungus with the strongest ammonification abilities: Z1 (bacteria) was obtained from fresh cow manure; Z2 (bacteria) was obtained from the thermophilic stage of cow manure composting; and F1 (fungus) was obtained from the maturation stage of cow manure composting.

[0031] Screening process: 10 g of cow dung samples from different sources were placed in Erlenmeyer flasks containing 0.9% physiological saline (1:10, w / v) and shaken for 2 h (30℃, 160 rpm / min) to prepare bacterial suspensions. Then, 10 mL of each bacterial suspension was placed in a 250 mL Erlenmeyer flask containing 100 mL of M1 and cultured at a constant temperature with shaking (30℃, 160 rpm / min) for 1 week. After 1 week, the culture was inoculated again into enrichment medium, and ammonification capacity was assessed using Nessler's reagent, repeated three times. 100 μL of bacterial suspension was extracted from each of the three enrichment media and inoculated into solid medium M1 using the dilution plating method. After incubation upside down at 30℃ for 1 week, single colonies with good growth were picked and repeatedly purified using the streak plate method to obtain three highly efficient ammonifying microorganisms (Z1, Z2, and F1). In addition, F1 was subjected to a three-month ammonia tolerance test (at 0.2 g / L, 0.4 g / L, 0.8 g / L and 1.6 g / L NH4, respectively). + (Domestication) to give it higher ammonification activity, which will provide strains for subsequent experimental research.

[0032] 2.2 Strain Identification DNA was extracted from strains Z1, Z2, and F1 using the Ezup column-based bacterial and fungal genomic DNA extraction kit. PCR amplification was performed using universal primers for bacterial 16S rDNA (F: 5'-AGAGTTGATCMTGGCTCAG-3', SEQ ID NO.1; R: 5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2) and ITS (F: 5'-TCCCGTAGGTGAACCTGCGG, SEQ ID NO.3; R: 5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO.4) sequences (PCR reaction system and conditions are shown in Tables 1 and 2). PCR products were sent to Sangon Biotech for sequencing and compared with the NCBI database. A phylogenetic tree was then constructed using MEGAversion 7.0 software.

[0033] Table 1 PCR reaction system Table 2 PCR reaction conditions 2.3. Strain Antagonism Experiment Single colonies of Z1, Z2 and F1, which had been purified multiple times, were inoculated into Landy solid medium and cultured at 30°C for 120 h (every 24 h). The antagonistic effect of the three strains was observed.

[0034] 2.4 Growth curves and Gram staining of the strain Bacterial (Z1 and Z2) growth curves: Bacteria Z1 and Z2 were inoculated into 100 mL of M1 liquid medium and cultured at a constant temperature with shaking (37℃, 160 rpm / min) for 60 h. During the culture period, the OD of the strains was measured every 6 h. 600 value.

[0035] Fungal (F1) growth curve: Fungal F1 was inoculated into PDA liquid medium and cultured at a constant temperature with shaking (30℃, 160 rpm / min) for one week. The bacterial solution was collected at regular intervals and in quantitative amounts every day. The collected bacterial solution was centrifuged (5000 r / min) for 5 min to collect the precipitate. The precipitate was dried in an oven at 60℃ overnight before collecting the biomass.

[0036] Gram staining of bacteria: Gram staining is performed on bacteria Z1, Z2, and F1 through smear preparation, fixation, primary staining, mordant staining, destaining, and microscopic examination.

[0037] 2.5 Optimal formulation of thermophilic ammonification compound microbial inoculant Based on the development method of high-efficiency thermophilic ammonification compound microbial agent for livestock manure composting: The colony count per unit volume of three microbial cultures at 80% of the logarithmic phase, and the viable cell count per unit volume of cultures of Z1, Z2, and F1 at 30℃ were determined. The inoculation volume ratio for preparing the novel nitrogen-preserving, thermophilic, ammoniated composite microbial agent (Amm-4) was calculated using the following formula.

[0038] The inoculation volume ratio of two types of ambient-temperature microorganisms = MZ1 / IZ1∶MF1 / IF1; the inoculation volume ratio of one type of thermophilic microorganism = MZ2 / IZ2; the ratio of the total volume of thermophilic microorganisms to the total volume of thermophilic microorganisms = [(MZ1+MF1) / (IZ1+IF1]:(MZ2 / IZ2); Wherein, MX is the number of colonies of type X bacteria in a single bacterial culture sample; IX is the number of colonies of this type of bacteria per unit volume in the bacterial culture at 80% of the logarithmic growth phase, expressed in CFU / mL (spores / mL).

[0039] The volume ratio of mesophilic microorganisms to thermophilic microorganisms is calculated as the ratio of the total number of viable bacteria in a unit mass sample to the total number of viable bacteria in a unit volume of bacterial solution.

[0040] Further colorimetric experiments using Nessler's reagent yielded the final thermophilic ammonifying compound microbial agent (Amm-4) inoculum volume ratio of *Bacillus fusiformis* (Z1): *Bacillus megaterium* (Z2): white-rot fungi (F1) = 1.2:1.6:1. It should be noted that the volume ratio of *Bacillus fusiformis* (Z1): *Bacillus megaterium* (Z2): white-rot fungi (F1) can vary within the range of 1.1~1.2:1.4~1.6:0.9~1, and the aforementioned volume ratio of 1.2:1.6:1 is not a specific limitation on the total volume of the mixture.

[0041] 2.6 Determination of strain domestication and bacterial liquid ammoniation ability at different temperatures Three single colonies (Z1, Z2, and F1) were inoculated into 100 mL of Landy liquid medium and cultured to the logarithmic growth phase. Then, the three bacterial cultures were mixed in 100 mL of sterilized Landy liquid medium according to the above mixing ratio to obtain four ammonifying microbial agents: Amm-1 (thermophilic fungus F1), Amm-2 (thermophilic bacterium Z1), Amm-3 (thermophilic bacterium Z2), and Amm-4 (Z1:Z1:F2 = 1.2:1.6:1). These were enriched four times under the same conditions (30℃, 160 rpm / min). Finally, the four enriched bacterial cultures were acclimatized sequentially at temperature gradients of 30℃, 40℃, 50℃, and 60℃ (incubated for 7 days at each temperature before being transferred to the next temperature gradient, from 30℃ to 60℃), for a total acclimatization period of 28 days. Meanwhile, the ammoniation capacity was first preliminarily tested using Nessler's reagent, and then a second quantitative identification was performed using an automated flow analyzer (AUTOAnalyzer 3) to determine the ammoniation capacity of the enriched bacterial solution at different temperatures.

[0042] 2.7 OD of bacteria during domestication 600 Biomass determination of fungi OD of bacteria (Z1 and Z2) cultured at different temperatures in logarithmic growth phase 600 The biomass of fungi was measured. Fungi (F1) were cultured on PDA liquid medium for 96 h (30℃, 160 rpm / min), centrifuged (5000 r / min) for 5 min to collect the precipitate, dried overnight in a 60℃ oven, and then the biomass was collected.

[0043] 2.8 Morphological identification of bacterial culture after high-temperature acclimatization Morphological changes of bacterial cultures Amm-1, Amm-2, Amm-3, and Amm-4 after acclimatization at 60 °C were observed using electron microscopy (SEM), and energy dispersive spectroscopy (EDS) was used to scan the co-cultured Amm-4.

[0044] 2.9 Preparation of Four Ammonia-Generating Microbial Agents The obtained enriched culture medium was centrifuged and resuspended. The bacterial and fungal suspensions were diluted separately with sterile distilled water to approximately 1×10⁻⁶. 8 cfu / mL and 1×10 8 spores / mL. A thermophilic ammonifying compound microbial inoculant (Amm-4) was obtained: (Z1:Z2:F1 = 1.2:1.6:1), which was used for subsequent composting experiments.

[0045] 2.10 Experimental Design for Inoculating Thermophilic Ammonifying Compound Microbial Agent (Amm-4) During Composting The inoculum amount of thermophilic ammonifying compound microbial agent (Amm-4) in cow manure compost was determined to be 5% (v / w) according to previous studies. The basic physicochemical properties of the compost materials are shown in Table 3. Wheat straw was used to adjust the C / N ratio and moisture content of the compost materials to approximately 30:1 and 65%, respectively, before thorough mixing. Cow manure composting was carried out in a 120 L fermenter with timely aeration at a rate of 0.4 L / kg / min. Five treatment groups were set up: Con (sterile distilled water) and Amm-4 (Z1:Z2:F1 = 1.2:1.6:1). The entire composting cycle was 42 days. Manual turning and sample collection were carried out on days 1, 3, 7, 14, 21, 28, 35, and 42. The collected compost samples were divided into two portions: one portion was used for physicochemical property analysis, and the other portion was stored at -80℃ for biological experiments.

[0046] Table 3 Composting materials used in this invention 2.11. Determination of indicators during composting after inoculation with thermophilic ammonifying compound microbial agent (Amm-4) Fermentation temperature of the compost pile was recorded daily. pH, conductivity, total Kjeldahl nitrogen (TNK), and organic matter content of the samples were determined according to previous laboratory methods. NH3 was collected using the boric acid method, once daily for the first two weeks, and at least three times weekly thereafter. The concentration of collected NH3 was determined by titration with H2SO4 (0.5 mol / L). Ammonium nitrogen (NH4) + -N) and nitrate nitrogen (NO3) - The determination of N₂O was performed using the previous method and a flow analyzer. N₂O was determined using a gas chromatograph. The sampling time and frequency for CO₂ and CH₄ were the same as those for NH₃, and the concentrations of the collected gases were determined using a gas chromatograph. Fresh compost samples were thoroughly mixed with sterile distilled water (1:10, w / v), and dissolved organic carbon and total organic carbon were determined according to the previous method.

[0047] Humic acid (HA) determination: Weigh 2 g of dry sample into a 50 mL centrifuge tube, add 20 mL of extraction solution (8 g NaOH + 89.212 g sodium pyrophosphate + 2 L distilled water), shake at 12000 r / min for 16 h, then centrifuge at 4000 r / min for 15 min, collect the supernatant into a centrifuge tube, add another 20 mL of extraction solution (8 g NaOH + 89.212 g sodium pyrophosphate + 2 L distilled water), repeat this process three times to collect the centrifuged liquid, then filter and perform vacuum filtration.

[0048] Amylase activity was measured at 540 nm using a microplate reader. Polyphenol oxidase activity was measured at 474 nm. Cellulase and laccase activities were measured at 420 nm and 540 nm, respectively, using previous methods. Calculations were performed as follows:

[0049] Note: V1: Total reaction volume; V2: Total sample solution volume; V3: Sample volume; A: Absorbance value; W: Sample weight; T: Total reaction time; ε: Absorption coefficient; D: Dilution factor; d: Optical path length.

[0050] 2.12 Evaluation of compost product quality after inoculation with thermophilic ammonifying compound microbial agent (Amm-4) Spectral characterization of compost product quality: An F-4600 microscope was used to record fluorescence intensity in the range of 250 nm to 600 nm at a scan rate of 1200 nm / min; the wavelength interval was 10 nm; and the reaction time was 0.1 s. The fluorescence intensity variation in the emission wavelength range of 300 nm to 480 nm at an excitation wavelength of 254 nm was recorded. The integral area ratio at 435 nm to 480 nm and 300 nm to 345 nm was denoted as A. 435~480 / A 300~345 The excitation wavelength of three-dimensional fluorescence spectroscopy (3D-EEM) is 200 nm to 440 nm, and the emission wavelength is 280 nm to 520 nm.

[0051] Pot experiment on vegetables using compost products: Poor soil was air-dried and sieved, then 2.5% of well-rotted compost products were added and mixed thoroughly. Each treatment was replicated in six places, with ten cabbage seeds sown in each pot. After 50 days of cultivation, ten healthy cabbage plants from each treatment were selected for analysis. Chlorophyll content was analyzed using a SPAD 502 chlorophyll analyzer.

[0052] 3. Results 3.1. Strains Isolation and PCR Identification PCR amplification was performed on the isolated Z1, Z2, and F1 samples. The electrophoresis results of the PCR amplification products are shown below. Figure 1 .

[0053] 3.2 Identification of key genes involved in ammoniation Urease gene ( UreC) It is widely recognized as a key gene in ammoniation. For example... Figure 2 The virus was detected in the genes of strains Z1, Z2, and F1. UreCThis further confirmed at the molecular level that all three strains have ammoniation activity and belong to ammonifying microorganisms.

[0054] 3.3 Construction of the phylogenetic tree of the strains The sequences of the PCR amplified products were compared with those in the NCBI gene database, and a phylogenetic tree was constructed for the top 10 most abundant strains. Figure 3 Strains Z1, Z2, and F1 were respectively Lysinibacillus fusiformis (Spindle-shaped Lysine Bacillus) Bacillus megaterium (Bacillus megaterium) and Phanerodontia chrysosporium (White rot fungus).

[0055] 3.4. Strain Antagonism Experiment like Figure 4 At 48h and 72h, strains Z1 and Z2 showed no antagonism, while the antagonistic relationship between Z1, Z2, and F1 was difficult to determine, possibly due to the slower growth of fungus F1. Upon re-observation after 120h, F1 completely covered the entire plate culture medium, completely covering Z1 and Z2. In conclusion, strains Z1, Z2, and F1 do not exhibit antagonism.

[0056] 3.5 Gram staining and morphological characteristics of the strain like Figure 5 Bacteria Z1 and Z2 were Gram-positive upon staining. Strain Z1 was relatively slender, with possibly rounded ends, and existed singly or in short chains. Strain Z2 was rod-shaped, with rounded ends, and existed singly or in short chains. Strain F1 consisted mainly of numerous hyphae and appeared white on the culture medium.

[0057] 3.6 Growth curve of the strain like Figure 6 During a 60-hour observation period, the growth curves of bacteria Z1 and Z2 were found to be roughly the same, with the logarithmic growth phase occurring between 12 and 24 hours. This is likely because both are Bacillus-like microorganisms, thus exhibiting some similar growth characteristics and trends. Furthermore, a 7-day growth curve analysis of fungus F1 revealed that the logarithmic growth phase of F1 occurred on day 4, and it essentially reached a plateau on day 5. Subsequently, based on the growth curves, four bacterial suspensions were prepared: Amm-1 (mesotropic fungus F1), Amm-2 (mesotropic bacterium Z1), Amm-3 (thermotropic bacterium Z2), and Amm-4 (Z1:Z2:F1 = 1.2:1.6:1).

[0058] 3.7 Determination of strain acclimatization and bacterial liquid ammonification ability at different temperatures like Figure 7As can be seen from the Nessler colorimetric reaction, at culture temperatures of 30℃ and 60℃, the degree of color development (ammoniation ability) of each bacterial culture is in the order of Amm-4>Amm-3>Amm-2>Amm-1>Control.

[0059] Subsequently, the ammonification capacity of each strain of bacteria acclimated at different temperatures was quantitatively detected. For example... Figure 8 In section a, the NH4 content of each bacterial culture + The yield of NH4+ in Amm-4 generally follows the order Amm-4 > Amm-3 > Amm-2 > Amm-1 at different temperatures, especially at 60℃. + The yield (204.83±6.73 mg / L) was significantly higher than that of other single-strain bacterial suspensions ( p <0.05). For example... Figure 8 In the middle b group, the biomass of Amm-1 gradually decreased with increasing temperature, showing a significant decrease at 60℃. The OD of Amm-2, Amm-3, and Amm-4... 600 The value decreases with increasing temperature; at 60℃, the OD of Amm-2... 600 The values ​​decreased significantly, while the OD values ​​of Amm-3 and Amm-4 decreased significantly. 600 The values ​​remain high, and Amm-4 > Amm-3. In summary, Amm-4 exhibits the highest ammoniation capacity at different temperatures and maintains high growth and ammoniation activity at 60℃.

[0060] 3.8 Scanning electron microscopy of bacterial culture after high-temperature acclimatization SEM results of the bacterial culture after acclimation at 60℃ are as follows: Figure 9 As shown, the Amm-1 bacterial culture formed a large number of dense biofilms and hyphal structures. After high-temperature acclimation, the Amm-2 and Amm-3 bacterial cultures did not show significant damage to bacterial morphology; the bacterial cell structure remained intact. The scanning electron microscopy results of Amm-4 showed that the biofilm produced by the fungi encapsulated a large number of bacteria, forming a stable structure. These results indicate that the morphology of the strains was not destroyed after high-temperature gradient acclimation, and the bacteria and fungi in the resulting thermophilic ammonifying composite microbial agent (Amm-4) formed a stable structure. This structure may increase the synergistic effect between strains, thereby enhancing the growth and ammonification performance of Amm-4. Amm-4 was determined to be the optimal ammonifying agent. Amm-4 will be used as the agent for subsequent experiments.

[0061] 3.9 Energy spectral scanning of thermophilic ammonification compound microbial agents like Figure 10Further energy dispersive spectroscopy (EDS) was performed on the thermophilic ammonification compound microbial agent (Amm-4). Sigma (σ) is a comprehensive indicator in EDS analysis, which includes both the percentage of elements in the mixture and reflects the confidence level of this proportion and the dispersion of the data distribution. The results of Sigma (σ) showed that N (0.34) > C (0.29) > O (0.07) in the mixed agent, which also reflects the high contribution of N element in the acclimated bacterial solution, which may also be due to the transport of N between strains.

[0062] 3.10. Effects of inoculating thermophilic ammonifying compound microbial agents on temperature and organic matter during composting. like Figure 11 In treatment a, as composting progressed, the highest temperatures in each treatment reached 58.8℃ (Con) and 68.0℃ (Amm⁻⁴), respectively. These temperatures were maintained for 7 days and 19 days, respectively, during the thermophilic phase (>55℃). Afterward, the temperatures in all treatments gradually decreased to ambient temperature. The effect of the microbial inoculum on fermentation temperature was Amm⁻⁴ > Con. Figure 11 b and Figure 11 The values ​​of pH, electrical conductivity (EC), and concentration all showed a consistent trend and met the maturity criteria, indicating that Amm-4 inoculation did not affect the composting process. Changes in organic matter (OM) content were as follows: Figure 11 As shown in Figure d, the organic matter content gradually decreased throughout the process. At the end of humification, the organic matter content in each treatment group was 65.64% (Con) and 60.55% (Amm-4), respectively, with corresponding organic matter degradation rates of 17.23% (Con) and 25.25% (Amm-4). During composting, the organic matter degradation rate in the Amm-4 inoculated group was higher than that in the control group.

[0063] 3.11. Amm-4 and its effect on NH4 in compost + -N, NO3 - -N, TKN and NH4 + -N / NO3 - The effect of -N NH4 + The change in -N reflects the degree of nitrogen transformation and ammoniation of organic matter. In all treatments, NH4... + -N all reached a rapid peak on day 3 of composting, and then gradually decreased until humification was completed. Figure 12 (a) NH4 in each treatment + The -N peak values ​​were 1.33 g / kg (Con) and 1.96 g / kg (Amm-4), respectively. At the end of humification, the NH4+ levels in each treatment were... +-N contents were 0.54 g / kg (Con) and 0.58 g / kg (Amm-4), respectively. NO3 in each treatment... - -N content changes as follows Figure 12 b. NO3 28 days before composting - The NO3 content is very low; as the material gradually decomposes, the NO3 content increases in each treatment process. - -N content increases rapidly and reaches its maximum at the end of humification, at 0.34 g / kg (Con) and 0.91 g / kg (Amm-4), respectively.

[0064] TKN content reflects the quality of compost humification products. Due to the rapid biodegradation of organic matter and high NH3 volatilization, the TKN content decreases significantly in the first 3 days ( Figure 12 The concentration of TKN in each treatment (con) increased significantly until it stabilized. TKN in all treatments peaked at the end of humification, at 22.85 g / kg (Con) and 28.42 g / kg (Amm⁻⁴), respectively. NH₄⁺ + -N / NO3 - -N can reflect the degree of compost decomposition ( Figure 12 (d). The highest ratio in the first 3 days is mainly related to NH4. + It is related to the formation of -N. At the end of humification, NH4 + -N / NO3 - -N values ​​were 1.57 (Con) and 0.63 (Amm-4), respectively. All treatments met the maturity standard (0.5~3.0), and the Amm-4 treatment group had a higher degree of maturity.

[0065] 3.12. Effects of Amm-4 inoculation on the release of NH3 and N2O during composting. like Figure 13 As shown in Figure a, the cumulative NH3 emissions were 10.3 g (Con) and 7.90 g (Amm⁻⁴), respectively. Compared with Con, the cumulative emissions of the treatment group decreased by 23.3% (Amm⁻⁴). In terms of cumulative N₂O emissions, the control group had the highest cumulative N₂O emissions. Figure 13 The concentrations of N2O inoculation (b) were 720.58 mg (Con) and 280.07 mg (Amm-4). Compared with the control group, Amm-4 inoculation significantly reduced N2O emissions by 61.1%, which may be because the inoculated Amm-4 promoted the nitrification process and inhibited denitrification during composting.

[0066] 3.13. Effects of Amm-4 inoculation on total organic carbon and dissolved organic carbon during composting. Due to the mineralization of organic carbon by microorganisms, the total organic carbon content decreases significantly as the composting and humification process proceeds.Figure 14 (a) The total organic carbon (TOC) in all treatments was 46%–46.98% in the initial stage. TOC gradually decreased as composting progressed, stabilizing at the end of humification. This downward trend was due to the rapid degradation of carbonaceous substances such as fats and carbohydrates by microorganisms and the mineralization of organic matter. At the end of humification, the TOC contents in Con and Amm-4 were 38.08% and 35.12%, respectively. Figure 14 In the first three days of composting, dissolved organic carbon (DOC) increased, followed by a rapid decline at 14 days. This is likely due to the microorganisms breaking down large amounts of solid polymers into substances such as CO2, releasing significant energy during the thermophilic phase to sustain their growth. At the end of humification, the DOC contents were 19.36% (Con) and 15.60% (Amm-4), respectively.

[0067] 3.14. The impact of Amm-4 inoculation during composting on the cumulative emissions of CO2 and CH4. like Figure 15 As shown, the cumulative CO2 emissions for each treatment were 2321.90 g (Con) and 1570.09 g (Amm-4), respectively. Compared to the control group, CO2 emissions decreased by 32.4% after Amm-4 inoculation. At the end of composting, the cumulative CH4 emissions for each treatment were 9.16 g (Con) and 7.51 g (Amm-4), respectively. Compared to the control group, CH4 emissions in the Amm-4 inoculated group decreased by 18.0%.

[0068] 3.15. Effects of Amm-4 inoculation during composting on polyphenol oxidase activity (a), amylase activity (b), laccase activity (c), and cellulase activity (d). Polyphenol oxidase activity ( Figure 16 a) and laccase activity ( Figure 16 The trends in (c) were similar, both initially increasing and peaking on day 21, then decreasing at the end of composting. This indicates that composting began to mature on day 21, with POA and LA causing a large amount of carbon-containing organic matter to condense into humus. At day 21, POA was 2013.36 nmol / h / g (Con) and 2321.34 nmol / h / g (Amm-4), respectively. At the end of composting, the POA in Amm-4 (1940.39 nmol / h / g) was significantly higher than that in the control group (1284.45 nmol / h / g). p<0.05). This may be because inoculation with Amm-4 can enhance ammoniation, causing nitrogenous proteins, amino acids, and other substances to condense with simple compounds produced by the oxidation of polyphenols (polycyclic aromatic hydrocarbons) and microbial metabolites to form humus. LA can promote lignin degradation; the LA levels at day 21 of composting were 25.34 mg / min / g (Con) and 28.32 mg / min / g (Amm-4), respectively. At the end of composting, compared to Con, the LA level in the Amm-4 inoculated group increased by 11.61%.

[0069] Amylase and cellulase activities peaked on day 7 and then gradually declined, indicating that starch substrates and cellulose undergo rapid degradation during the thermophilic phase. At day 7, compared to Con, the AA content in the Amm-4 inoculated group increased by 29.91% ( Figure 16 (b) CA increased by 30.43% ( Figure 16 (d). At the end of composting, Amm-4 had 10.25% more AA than Con ( p <0.05). However, CA levels did not differ significantly among treatments at the end of composting. The results indicate that AA and CA in the Amm-4 inoculated group were significantly higher than in the control group during the thermophilic phase. This suggests that the thermophilic ammonifying compound microbial agent can enhance enzyme activity in compost.

[0070] 3.16. Effect of Amm-4 inoculation on the degree of humification during composting. Three-dimensional fluorescence spectroscopy (3D-EEM) can characterize the degree of humification of compost products. Each substance has a specific fluorescent group, and changes in the substance can be characterized according to specific wavelength regions (I, II, III, IV, V) (Table 4). In the initial material ( Figure 18 The protein content (such as tryptophan) in region a) was significantly higher than that in the final product at the end of composting. Figure 18 (b and c) This may be due to the degradation of unstable macromolecules and the formation of humic substances during the composting and humification process. The decrease in region IV during composting may be due to the reuse of soluble microbial metabolites by microorganisms. Compared to the initial material, regions III and V in the compost product are significantly increased, possibly due to the formation of a large amount of humic matter at the end of composting, resulting in stronger fluorescent groups in 3D-EEM characterization. Furthermore, compared to the Con and Amm-4 treatment groups, the fluorescence regions and intensities of regions III and V are larger ( Figure 18 (c) This may be because Amm-4 inoculation promoted the composting humification process and humus formation. More precise quantitative analysis was performed on the five regions of the 3D-EEM based on fluorescence region integration. Figure 18(d). The percentage of each fluorescent group (Pi, n) after MATLAB treatment can more intuitively reveal the proportion of substances. In region V, compared with Con, the humification area of ​​the Amm-4 treatment group increased significantly by 35%.

[0071] Table 4. Wavelength regions of 3D-EEM fluorescence spectra (nm) 3.17. Effect of Amm-4 inoculation during composting on compost product quality (pot experiment) Pot experiments can be used to assess the quality of compost products. Figure 17 Compared with Con, the Amm-4 inoculated group showed significant increases in chlorophyll content, plant height, root length, and biomass of Chinese cabbage, respectively, by 36.76%, 98.18%, 52.62%, and 84.04%. Figure 17 (a~d). This indicates that compost products inoculated with Amm-4 are more conducive to plant root development and promote nutrient accumulation. The higher chlorophyll content also proves that compost products inoculated with Amm-4 are beneficial to plant chlorophyll synthesis, directly affecting plant photosynthesis and thus promoting plant growth. In conclusion, Amm-4 inoculation improves the quality of compost products and is beneficial to crop growth.

[0072] Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A thermophilic ammonifying compound microbial agent, characterized in that, It is composed of spindle-shaped lysine-containing Bacillus ( Lysinibacillus fusiformis ), Bacillus megaterium ( Bacillus megaterium It is a compound of Bacillus fusiformis and Phanerochaete chrysosporium; the accession number of Bacillus fusiformis is CCTCC NO: M 20242776, the accession number of Bacillus megaterium is CCTCC NO: M 20242777, and the accession number of Phanerochaete chrysosporium is CCTCC NO: M 20242778. All three strains are deposited at the China Center for Type Culture Collection.

2. The thermophilic ammonifying compound microbial agent according to claim 1, characterized in that, The volume ratio of *Bacillus spindleii*, *Bacillus megaterium*, and *white-rot fungi* inoculum was 1.1–1.2:1.4–1.6:0.9–1, and the effective viable count of the thermophilic ammonifying composite microbial agent was 1 × 10⁻⁶. 8 ~10 9 cfu / mL.

3. The thermophilic ammonifying compound microbial agent according to claim 1, characterized in that, The thermophilic ammonifying compound microbial agent is a compound of Bacillus spindleii, Bacillus megaterium, white-rot fungi, and microbiologically acceptable excipients.

4. A method for preparing the thermophilic ammonifying composite microbial agent according to claim 1, characterized in that, Includes the following steps: After activating Bacillus fusiformis, Bacillus megaterium, and white-rot fungi respectively, they were inoculated into Landy liquid medium and cultured together to obtain the thermophilic ammonifying compound microbial agent.

5. The application of the thermophilic ammonifying compound microbial agent according to any one of claims 1 to 3 in composting.

6. The application according to claim 5, characterized in that, The thermophilic ammonification compound microbial agent is used to promote the ammonification process.

7. The application according to claim 5, characterized in that, The thermophilic ammonification compound microbial agent is used to promote the composting process and improve the quality of compost products.

8. A fertilizer, characterized in that, It is obtained by inoculating the thermophilic ammonifying compound microbial agent according to any one of claims 1 to 3 into compost material for composting fermentation.

9. Use of the fertilizer of claim 8 in promoting plant growth.

10. The use according to claim 9, characterized in that, The plant in question is Chinese cabbage.

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