Lysinibacillus fusiformis, fermentation liquor, ammonifying bacterium agent and application

Abstract

The invention belongs to the field of resource utilization of livestock and poultry manure, and particularly relates to a spindle-shaped lysine bacillus, fermentation liquor, an ammonifying bacterium agent and application. The lysinibacillus fusiforme is preserved in the China Center for Type Culture Collection, and the preservation number of the lysinibacillus fusiforme is CCTCC (China Center for Type Culture Collection) NO: M 20242776. Tests prove that the ammoniating bacterium inoculant prepared by utilizing the lysine bacillus spunlaginosus shows higher ammoniating activity at 37 DEG C, and the ammoniating bacterium inoculant can promote the composting recycling process and improve the composting product quality by inoculating the ammoniating bacterium inoculant.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of livestock manure resource utilization, and particularly relates to a fusiform Bacillus lysiniformis, a fermentation liquor, an ammonification bacterial agent and a use thereof in a compost resource utilization process. BACKGROUND

[0002] Livestock manure has an annual output of more than 3 billion tons, and has the dual nature of pollution and resources. Livestock manure resource utilization is an effective means to address the problems of livestock manure pollution and resource waste. With the rapid development of intensive farming, the output of livestock manure has increased dramatically, causing serious pollution to the environment and leading to the spread of pathogenic bacteria. Therefore, converting livestock manure into valuable resources not only can improve the breeding environment and ecological environment pollution, but also can improve the utilization efficiency of livestock manure resources, so that livestock manure becomes a high-value product. Composting technology is one of the important ways to utilize livestock manure resources and harmlessly, which can convert easily degradable organic matter into high-value compost products and organic fertilizers 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 manure, composting has the advantages of short cycle, simple process flow, small land occupation, low investment cost, mature technology, etc., and has a broad development prospect and market demand, and is widely used in livestock manure resource utilization and harmless treatment. In addition, composting can convert livestock manure into stable, sanitary and high-nutrient organic fertilizers 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 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 Bacillus amyloliquefaciens is often used to degrade proteins in diatomite, and in related reports of composting, bacteria (Bacillus subtilis, Bacillus amyloliquefaciens, etc.) have been proven to have high ammonification activity. Bacillus and Pseudomonas

[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 volatile ammonia (NH3) and ammonium (NH4 + ​) formation, which is also an important step of nitrogen transformation, ammonifying microorganisms determine the transfer balance between the redox reactions related to nitrogen. Ammonifying microorganisms play a key limiting role in nitrogen transformation microorganisms. Ammonifying microorganisms degrade nitrogen-containing compounds (urea, amino acids, nicotine, etc.) into small-molecule organic nitrogen that can be absorbed and utilized, and then biological utilization (decomposition and synthesis) is carried out. Most microorganisms exhibit ammonification activity, including bacteria and fungi. For example, Proteobacteria and Firmicutes contain members that are extremely important for global carbon and nitrogen cycles, such as Pseudomonas and Bacillus, which are widely distributed and have high ammonification activity. In addition, not only can ammonifying microorganisms rapidly start the fermentation process, generate heat through their metabolic activity, and increase the temperature of the pile to accelerate the reproduction and activity of other microorganisms, but the activity of ammonifying microorganisms promotes the transformation and fixation of nitrogen in the compost, providing a material basis for the formation of high-value compost products.

[0005] Compared with ammonifying fungi, ammonifying bacteria have the ability to reproduce rapidly and utilize organic matter (including starch and protein), and are mainly responsible for the synthesis and decomposition of substances in the thermophilic stage, so they have attracted the attention of researchers. In the initial stage of composting, bacteria can utilize easily degradable organic matter to complete their own proliferation and dominate in the thermophilic stage. For example, Bacillus can produce endospores or thick-walled spores to withstand adverse environments such as high temperature, and also plays a role in the degradation of starch and protein. A large number of studies have also shown that inoculation of nitrogen transformation bacterial inoculants has many benefits in the composting process. For example, inoculation of ammonia-oxidizing bacteria can facilitate the conversion of NH4 + -N to NO2 - -N, and inoculation of nitrifying bacteria has a positive effect on the accumulation of NO3 - -N and the nitrogen fixation process. In addition, NH4 + -N can also be fixed as biological nitrogen in organic matter by ammonia-assimilating bacteria. However, there are few reports on the practical application of ammonifying bacterial inoculants prepared from a single strain in the composting of livestock and poultry manure resources, but the preparation of ammonifying bacterial inoculants will provide a driving force for the composting process. Therefore, the present application intends to provide a new ammonifying bacterium to promote the composting resourceization process and improve the quality of compost products. SUMMARY

[0006] Based on the above technical problems, the present application provides a fusiform lysine Bacillus, which is prepared into an ammonifying bacterial inoculant, and the ammonifying bacterial inoculant has better ammonification capacity and composting effect.

[0007] The specific technical solutions provided by the present application are as follows: In a first aspect, the present application provides a fusiform lysine Bacillus (Bacillus fusiformis lysine), Lysinibacillus fusiformis which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20242776.

[0008] In a second aspect of the present application, a fermentation method of the Bacillus licheniformis is provided, comprising the following steps: Inoculating the Bacillus licheniformis into an ammonification-rich culture medium and culturing at 32-37°C for 3-7 days.

[0009] In a third aspect of the present application, a fermentation broth obtained by the above method is provided.

[0010] In a fourth aspect of the present application, an ammonification bacterial agent is provided, which takes the Bacillus licheniformis or the fermentation broth as an active ingredient.

[0011] As a preferred embodiment of the present application, the ammonification bacterial agent is compounded by the Bacillus licheniformis or the fermentation broth and a microbiologically acceptable excipient.

[0012] In a fifth aspect of the present application, the Bacillus licheniformis or the fermentation broth or the ammonification bacterial agent is applied in composting.

[0013] As a preferred embodiment of the present application, the ammonification bacterial agent or the fermentation broth or the ammonification bacterial agent is used to promote the ammonification process.

[0014] As a preferred embodiment of the present application, the ammonification bacterial agent or the fermentation broth or the ammonification bacterial agent is used to promote the composting of cow dung and improve the quality of composting products.

[0015] In a sixth aspect of the present application, a fertilizer is provided, which is obtained by inoculating the Bacillus licheniformis or the fermentation broth or the ammonification bacterial agent into composting materials for composting fermentation.

[0016] As a preferred embodiment of the present application, the composting materials are biomass waste or livestock excrement. For example, the biomass waste is straw, and the livestock excrement is cow dung.

[0017] In a seventh aspect of the present application, the fertilizer is applied in promoting the growth of plants, which is used to promote the accumulation of chlorophyll and biomass in plants, or to promote the growth of plant height and root length.

[0018] As a preferred embodiment of the present application, the plants are Chinese cabbage.

[0019] Compared with the prior art, the present application has the following beneficial effects: The present application separates and identifies a fusiform lysine bacillus, and the ammonification bacterial agent (Amm-2) prepared by using the fusiform lysine bacillus can promote the resource utilization process of compost and improve the quality of compost products. The test proves that the ammonification bacterial agent (Amm-2) shows high ammonification activity (NH4 + : 294.89±7.63 mg / L) at 37℃. Compared with the control group, inoculating Amm-2 in the process of cow manure composting can reduce 9.1% of NH3 and 40.0% of N2O emissions, increase the content of total Kjeldahl nitrogen (TKN), promote the degradation of total organic carbon (TOC) and dissolved organic carbon (DOC) in the process of cow manure composting, reduce 12.3% of CH4 emissions, and enhance the activity of carbon conversion related enzymes, and improve the humification degree of compost products. And through the pot experiment to evaluate the quality of compost products, the results show that compared with the control group, the compost products treated by Amm-2 increase the chlorophyll, plant height, root length and biomass of planted Chinese cabbage.

[0020] Biological material preservation information: The fusiform lysine bacillus has Latin name Lysinibacillus fusiformis Z1, and the taxonomic name is fusiform lysine bacillus Z1, the preservation number is CCTCC NO: M 20242776, the preservation date is December 10, 2024, the strain is preserved in China Typical Culture Collection Center, and the preservation address is Wuhan, Wuhan University, China. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is the PCR identification result of bacteria Z1; Figure 2 is the identification of the key ammonification gene of bacteria Z1, urease gene (ureC); UreC Figure 3 is the neighbor-joining phylogenetic tree based on gene sequence, which shows that strain Z1 is fusiform lysine bacillus; Figure 4 is the morphological observation result of bacteria Z1; Figure 5 is the growth curve of bacteria Z1; Figure 6 is the Nessler color reaction of ammonification bacterial agent (Amm-2) at 37℃ culture temperature; Figure 7 is the scanning electron microscope result of ammonification bacterial agent (Amm-2), 3μm; Figure 8 is the influence of inoculating ammonification bacterial agent (Amm-2) on the composting process during composting, a, temperature, b, pH, c, conductivity, d, organic matter content; ​Figure 9 Effect of inoculation of Amm-2 on NH4 + -N(a), NO3 − -N(b), TKN(c) and NH4 + -N / NO3 − -N(d) during composting process; Figure 10 Effect of inoculation of Amm-2 on cumulative NH3 emission (a) and cumulative N2O emission (b) during composting process; Figure 11 Effect of inoculation of Amm-2 on total organic carbon (a) and dissolved organic carbon (b) during composting process; Figure 12 Effect of inoculation of Amm-2 on cumulative CH4 emission during composting process; different letters in the figure represent significant difference; Figure 13 Effect of inoculation of Amm-2 on polyphenol oxidase activity (a), amylase activity (b), laccase activity (c) and cellulase activity (d) during composting process; Figure 14 Effect of inoculation of Amm-2 on humification degree during composting process, a, first day of composting, b, 42nd day of composting of the control group, c, 42nd day of composting of the Amm-2 treatment group, d, fluorescence area integration; Figure 15 Effect of inoculation of Amm-2 on compost product quality (pot experiment) during composting process, a, chlorophyll, b, plant height, c, root length, d, biomass. DETAILED DESCRIPTION

[0022] The technical solutions described in the present application will be further described below in conjunction with specific embodiments, but the present application is not limited thereto.

[0023] 1. Main reagents Bacterial culture medium (LB): beef extract 3.0 g / L, protein peptone 10.0 g / L, NaCl 5.0 g / L, agar 10 g / L and sterile distilled water 1.0 L, pH 7.2~7.4.

[0024] Ammonification enrichment medium (M1): protein peptone 5 g / L, KH2PO4 0.5 g / L, K2HPO4 0.2 g / L, MgSO4·7H2O 0.5 g / L, agar 15 g / L and sterile distilled water 1.0 L, pH 7.2.

[0025] Landy medium: glucose 20.0 g, yeast powder 1.0 g, L-glutamic acid 5.0 g, L-phenylalanine 2 mg, L-tryptophan 1.0 g, potassium chloride 0.5 g, potassium dihydrogen phosphate 1.0 g, magnesium sulfate heptahydrate 0.5 g, manganese sulfate tetrahydrate 5 mg, copper sulfate heptahydrate 0.16 mg, ferrous sulfate heptahydrate 0.15 mg, and 1.0 L of sterile distilled water.

[0026] 2. Method 2.1. Isolation and purification of strains Strain source: The strains were isolated from the cow dung samples from Ningxia Jingtian Ranch and different cow farms in Shaanxi using ammonification enrichment medium (M1). The present application initially screened 15 bacteria from different cow dung samples using enrichment culture method. The 15 bacterial strains were derived from fresh cow dung (excreted within 5 minutes, 30°C-37°C). The strongest ammonification ability was determined by the color reaction of Nessler reagent, and a strain Z1 (bacteria) with the strongest ammonification ability was obtained from the composting mature stage of cow dung.

[0027] Screening process: 10 g of samples from different sources were respectively placed in a triangular flask containing 0.9% physiological saline (1:10, w / v) and shaken for 2 h (37°C, 160 rpm / min) to prepare a bacterial suspension. Then, 10 mL of the bacterial suspension was respectively placed in a 250 mL conical flask containing 100 mL of M1 and subjected to constant temperature shaking (37°C, 160 rpm / min) for 1 week. After 1 week, the bacterial suspension was again inoculated into the primary enrichment medium, and the ammonification ability was identified by Nessler reagent. The process was repeated three times. Then, 100 μL of the bacterial suspension was extracted from the enrichment medium after three times of enrichment, and the dilution spread plate method was used to inoculate the solid medium M1. After 1 week of culture in a 37°C incubator, single colonies with better growth were picked, and the plate streaking method was used to repeatedly purify the single colonies to obtain a high-efficiency ammonification bacterium Z1 for subsequent experimental research.

[0028] 2.2. Strain identification The DNA of the strain Z1 was extracted by using Ezup column bacterial genome DNA extraction kit, and the universal primer bacterial 16S rDNA (F: 5'-AGAGTTTGATCMTGGCTCAG-3', SEQ ID NO.1; R: 5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2) sequence was subjected to PCR amplification (PCR reaction system and conditions as shown in Table 1 and Table 2). The PCR product was sent to Shengong for sequence determination and comparison in NCBI database. Subsequently, the MEGA version 7.0 software was used to construct a phylogenetic tree.

[0029] Table 1 PCR reaction system Table 2 PCR reaction conditions 2.3, Growth curve and Gram staining of the strain Bacterial (Z1) growth curve: Bacterial Z1 was inoculated into 100 mL M1 liquid medium and incubated at constant temperature (37°C, 160 rpm / min) for 60 h. During the incubation period, the OD value of the strain was determined every 6 h. 600

[0030] Bacterial Gram staining: Gram staining identification of bacterial Z1 was performed by smearing, fixing, primary staining, medium staining, decolorizing and microscopic examination.

[0031] 2.4, Strain domestication at different temperatures and determination of ammonia-oxidizing ability of bacterial liquid Single colony Z1 was inoculated into 100 mL Landy liquid medium, and after incubation to the logarithmic growth phase, the bacterial liquid was inoculated into sterilized 100 mL Landy liquid medium for mixed culture to obtain ammonia-oxidizing bacterial inoculant, marked as Amm-2, and enriched for 4 times under the same conditions (37°C, 160 rpm / min). Finally, the enriched bacterial liquid was domesticated at 30°C, 40°C, 50°C and 60°C (after 7 days of incubation at each temperature, it was transferred to the next temperature gradient, from 37°C to 60°C in turn), and the total domestication process lasted for 28 days. At the same time, preliminary detection of ammonia-oxidizing ability was performed using Nessler reagent, and secondary quantitative identification was performed using an automatic flow analyzer (AUTO Analyzer 3) to determine the ammonia-oxidizing ability of the enriched bacterial liquid at different temperatures.

[0032] 2.5, OD of bacteria during domestication 600 The OD value of bacterial Z1 in the logarithmic growth phase cultured at different temperatures was determined after being cultured in LB liquid medium for 24 h (37°C, 160 rpm / min). 600

[0033] 2.6, Morphological identification of bacterial liquid after high-temperature domestication The morphological structure of bacterial liquid Amm-2 after 60°C domestication was observed by electron microscope (SEM).

[0034] 2.7, Preparation of ammonia-oxidizing bacterial inoculant The enriched culture liquid obtained above was centrifuged, resuspended, and diluted with sterile distilled water to about 1x10 8 cfu / mL, which was used for subsequent composting experiments.

[0035] ​​2.8、Inoculation of ammoniating bacteria agent (Amm-2) in composting process The inoculation amount of ammoniating bacteria agent (Amm-2) in cow manure composting was determined as 5% (v / w) according to previous studies. The basic physicochemical properties of the composting material are shown in Table 3. After adjusting the C / N ratio and moisture content of the composting raw material to about 30:1 and 65% respectively using wheat straw, the materials were thoroughly mixed. The cow manure composting was carried out in a fermentation tank with a capacity of 120 L, and aeration was performed in time with an aeration rate of 0.4 L / kg / min. Two treatment groups were set up in the experiment: Con (sterile distilled water) and Amm-2. The entire composting period was 42 days, and artificial turning was performed on days 1, 3, 7, 14, 21, 28, 35, and 42, and samples were collected. The collected compost samples were divided into two parts, one for analysis of physicochemical properties and the other stored in a -80 ℃ refrigerator for biological experiments.

[0036] Table 3 Composting materials used in the present application 2.9、Measurement of indicators during composting after inoculation of ammoniating bacteria agent (Amm-2) The fermentation temperature of the compost was recorded daily. The pH, electric conductivity (EC), total Kjeldahl nitrogen (TKN), and organic matter (OM) of the samples were determined according to previous methods in the laboratory. NH3 was collected using the boric acid method, with a frequency of once a day for the first two weeks and at least three times a week later. The concentration of NH3 after collection was determined using the H2SO4 (0.5 mol / L) titration method. Ammonium nitrogen (NH4 + -N) and nitrate nitrogen (NO3 -Determination of N2O was performed using a gas chromatograph (Agilent Technologies 7890B Network GC system, America). CO2 and CH4 were collected for the same time and frequency as NH3, and the collected gas was measured for concentration using a gas chromatograph (Agilent Technologies 7890B Network GC system, America). Fresh compost samples were mixed thoroughly with sterile distilled water (1:10, w / v), and dissolved organic carbon (DOC) and total organic carbon (TOC) were determined according to previous methods (Xu et al. 2022b).

[0037] Humic acid (HA) determination: 2 g of dry sample was weighed into a 50 mL centrifuge tube, 20 mL of extraction solution (8 g NaOH + sodium pyrophosphate 89.212 g + 2 L distilled water) was added, and it was shaken at 12000 r / min for 16 h, then centrifuged at 4000 r / min for 15 min, the supernatant was taken into a centrifuge tube, 20 mL of extraction solution (8 g NaOH + sodium pyrophosphate 89.212 g + 2 L distilled water) was added again, and this was repeated three times to collect the centrifugal liquid, then filtered and suction filtered.

[0038] Amylase activity determination used a microplate reader to determine its activity at 540 nm. Polyphenol oxidase activity was determined at 474 nm. Cellulase activity and laccase activity were determined at 420 nm and 540 nm, respectively, using previous methods. The following method was used to calculate:

[0039] Note: V1: total reaction volume; V2: total volume of sample solution; V3: sample volume; A: absorbance; W: sample weight; T: total reaction time; ε: absorbance coefficient; D: dilution factor; d: light path.

[0040] 2.10, Evaluation of compost product quality after inoculation of ammonifying bacterial inoculant (Amm-2) The spectrum of the quality of the compost product was recorded by F-4600 (Hitachi manufacturing co. LTD, Japan) at 250 nm~600 nm, the scanning speed was 1200 nm / min, the wavelength interval was 10 nm, and the reaction was 0.1 s. When the excitation wavelength was 254 nm, the change of the fluorescence intensity at the emission wavelength of 300 nm~480 nm was recorded, and the integral area ratio at 435 nm~480 nm and 300 nm~345 nm was recorded as A 435~480 / A 300~345 The excitation wavelength of the three-dimensional fluorescence spectrum (3D-EEM) was 200 nm~440 nm, and the emission wavelength was 280 nm~520 nm.

[0041] The vegetable pot experiment of the compost product: the dry and sieved poor soil was mixed with 2.5% of the composting product, 6 repetitions were set for each treatment, 10 cabbage seeds were sown in each pot, and after 50 days of culture, ten well-grown cabbages were selected for detection in each treatment. The chlorophyll was analyzed by chlorophyll instrument SPAD 502.

[0042] 3、Results 3.1, Strain isolation and PCR identification The PCR product of the isolated Z1 was amplified and electrophoresed, as shown in Figure 1 .

[0043] 3.2, Identification of key genes of ammonification The urease gene ( UreC ) is recognized as a key gene of ammonification. As Figure 2 , the UreC was detected in the strain Z1 gene, which further confirmed from the molecular level that the strain Z1 had ammonification and belonged to ammonification microorganisms.

[0044] 3.3, Construction of phylogenetic tree of strain The sequence of the PCR amplified product was compared in the NCBI gene library, and the phylogenetic tree of the strain with high abundance in TOP10 was constructed Figure 3 ). The strain Z1 was identified as Lysinibacillus fusiformis (spindle-shaped lysine bacillus).

[0045] 3.4, Gram staining and morphological characteristics of the strain As Figure 4 , the Gram staining result of the bacteria Z1 was Gram-positive bacteria. The strain Z1 was relatively long and thin, the end might be round, and existed alone or formed short chain arrangement.

[0046] 3.5, Growth curve of the strain AsFigure 5 In the 60 h continuous observation, the logarithmic growth phase of bacteria Z1 was found to be during 12-24 h, which may be due to its being a Bacillus sp.

[0047] 3.6, Strain acclimation at different temperatures and determination of ammonia capacity of bacterial liquid As Figure 6 From the Nessler color reaction, it can be seen that at a culture temperature of 37°C, the color development of the bacterial liquid showed Amm-2 > Control.

[0048] Subsequently, the ammonia capacity of the bacterial liquid of the strain acclimated at different temperatures was further quantitatively detected. As shown in Table 4, the ammonia capacity and biomass of Amm-2 gradually decreased with the increase of temperature, and had the highest ammonia capacity (NH4 + : 294.89 ± 7.63 mg / L) and growth activity at 37°C.

[0049] Table 4 Determination of ammonia performance of Amm-2 at different temperatures using Nesler reagent and AUTO Analyzer 3 Note: The experiment was performed in triplicate, the inoculum was 10% (v / v), and the culture was performed in a 250 mL conical flask with 100 mL sterile medium. Fresh seed medium was cultured at 37°C for 7 days, then transferred to 40°C for 7 days, and then sequentially cultured at 50°C and 60°C. The acclimation process lasted for 28 days, a total of 48 times

[0050] 3.7, Scanning electron microscopy of bacterial liquid after high-temperature acclimation The SEM results of the bacterial liquid acclimated at 60°C are shown in Figure 7 After high-temperature acclimation, the Amm-2 bacterial liquid did not have obvious damage to the bacterial morphology, and the bacterial morphology and structure remained intact. This indicates that the strain has strong adaptability to high temperature.

[0051] 3.8, Effect of inoculating thermophilic ammonia bacteria agent (Amm-2) on temperature and organic matter during composting As Figure 8 In a, with the progress of composting, the highest temperature in each treatment reached 58.8°C (Con) and 63.5°C (Amm-2), respectively. And in the thermophilic stage (> 55°C), they maintained for 7 d and 10 d, respectively. After that, the temperature of all treatments gradually decreased to ambient temperature. The effect of microbial inoculant on fermentation temperature was Amm-2 > Con. As Figure 8 b and Figure 8The values ​​of pH and conductivity all showed a consistent trend and met the maturity standards, indicating that Amm-2 inoculation did not affect the composting process. Changes in organic matter content were as follows: Figure 8 As shown in Figure d, the organic matter content gradually decreased throughout the process. At the end of composting, the organic matter degradation rates in each treatment group were 17.23% (Con) and 20.22% (Amm-2), respectively. During composting, the organic matter degradation rate in the Amm-2 inoculated group was higher than that in the control group.

[0052] 3.9. Inoculate NH4+ with Amm-2 during composting. + -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 the end of composting. Figure 9 (a) NH4 in each treatment + The NO3- peak values ​​were 1.33 g / kg (Con) and 1.78 g / kg (Amm-2), respectively. NO3- in each treatment... - -N content changes as follows Figure 9 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. - The -N content increases rapidly and reaches its maximum at the end of composting, at 0.34 g / kg (Con) and 0.46 g / kg (Amm-2), respectively.

[0053] 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 9 The concentration of TKN in each treatment (con) increased significantly until it stabilized. TKN in all treatments peaked at the end of composting, at 22.85 g / kg (Con) and 27.04 g / kg (Amm⁻²), respectively. NH₄⁺ + -N / NO3 - -N can reflect the degree of compost decomposition ( Figure 9 (d). The highest ratio in the first 3 days is mainly related to NH4. + The formation of -N is related. At the end of composting, NH4... + -N / NO3 --N values ​​were 1.57 (Con) and 1.30 (Amm-2), respectively. All treatments met the maturity standard (0.5~3.0), and the Amm-2 treatment group had a higher degree of maturity.

[0054] 3.10. Effects of Amm-2 inoculation on the release of NH3 and N2O during composting. like Figure 10 As shown in Figure a, the cumulative NH3 emissions were 10.3 g (Con) and 9.36 g (Amm⁻²), respectively. Compared with the control group, the cumulative emissions of the treatment group decreased by 9.1% (Amm⁻²). In terms of cumulative N₂O emissions, the control group had the highest cumulative N₂O emissions. Figure 10 In the control group (b), the concentrations were 720.58 mg (Con) and 433.04 mg (Amm-2). Compared with the control group, Amm-2 inoculation significantly reduced N2O emissions by 40.0%, which may be because the inoculated Amm-2 promoted the nitrification process and inhibited denitrification during composting.

[0055] 3.11 Effects of Amm-2 inoculation during composting on total organic carbon (a) and dissolved organic carbon (b) Due to the mineralization of organic carbon by microorganisms, the total organic carbon (TOC) content decreases significantly as the composting and humification process progresses. Figure 11 (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 composting. 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 composting, the TOC content in Con and Amm-2 was 38.08% and 37.02%, respectively. Figure 11 In section b, the dissolved organic carbon (DOC) content increased during the first 3 days of composting, 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 a significant amount of energy during the thermophilic phase to sustain their growth activities. At the end of composting, the DOC content was 19.36% (Con) and 16.79% (Amm-2), respectively.

[0056] 3.12. The effect of Amm-2 inoculation during composting on cumulative CH4 emissions. like Figure 12 At the end of composting, the cumulative CH4 emissions for each treatment were 9.16 g (Con) and 8.03 g (Amm-2), respectively. Compared with the control group, the CH4 emissions in the Amm-2 inoculated group were reduced by 12.3% (Amm-2).

[0057] 3.13 Effects of Amm-2 inoculation during composting on polyphenol oxidase activity (a), amylase activity (b), laccase activity (c), and cellulase activity (d). Polyphenol oxidase activity (POA) Figure 13 a) and laccase activity (LA) Figure 13 c) showed the same trend, initially increasing and peaking on day 21, then decreasing at the end of composting. This indicates that composting begins to mature on day 21, with POA and LA causing a large amount of carbonaceous organic matter to condense into humus. POA at day 21 was 2013.36 nmol·h⁻¹. -1 ·g -1 (Con) and 2145.56 nmol·h -1 ·g -1 (Amm-2). At the end of composting, the POA of Amm-2 was significantly higher than that of the control group (1284.45 nmol·h⁻¹). -1 ·g -1 () p <0.05). This may be because inoculation with Amm-2 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 concentration of LA on day 21 of composting was 25.34 mg·min⁻¹. -1 ·g -1 (Con) and 26.75 mg·min -1 ·g -1 (Amm-2). At the end of composting, LA increased by 7.02% in the Amm-2 inoculated group compared to the control group.

[0058] Amylase activity (AA) and cellulase activity (CA) peaked on day 7 and then gradually declined, indicating that starch substrate and cellulose undergo rapid degradation during the thermophilic phase. At day 7, compared with the control group, the AA activity in the Amm-2 inoculated group was significantly lower. Figure 13 (b) and CA ( Figure 13 (d) increased. At the end of composting, the AA content of Amm-2 was 10.56% higher than that of Con. p <0.05. However, CA did not differ significantly among treatments at the end of composting ( p>0.05). The results showed that AA and CA in the Amm-2 inoculated group were significantly higher than those in the control group during the thermophilic phase. This indicates that the ammonifying bacteria inoculant (Amm-2) can enhance enzyme activity in compost.

[0059] 3.14. Effect of Amm-2 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 5). In the initial material ( Figure 14 The protein content (such as tryptophan) in region a) was significantly higher than that in the final product at the end of composting. Figure 14 (b and c) This may be due to the degradation of unstable macromolecular substances and the formation of humic substances during the composting process. As composting progresses, the decrease in region IV may be due to the reuse of soluble microbial metabolites by microorganisms. Compared to the initial material, regions III and V in the humified 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, at the end of composting, compared to the control group, the Amm-2 treatment group showed larger regions and greater intensities of fluorescence in regions III and V (…). Figure 14 (c) This may be because Amm-2 inoculation promoted the composting process and humus formation. More precise quantitative analysis of the five regions in the 3D-EEM was performed based on fluorescence region integration. Figure 14 (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 the control group, the humification area of ​​the Amm-2 treatment group increased significantly by 10%.

[0060] Table 5. Wavelength regions of 3D-EEM fluorescence spectra (nm) 3.15. Effect of Amm-2 inoculation during composting on compost product quality (pot experiment) Pot experiments can be used to assess the quality of compost products. Figure 15 Compared with the control group, the chlorophyll content, plant height, root length, and biomass of Chinese cabbage increased significantly by 12.32%, 46.78%, 40.49%, and 62.96%, respectively. Figure 15 (a~d). This indicates that compost products inoculated with Amm-2 are more conducive to plant root development and promote nutrient accumulation. The higher chlorophyll content also proves that compost products inoculated with Amm-2 are beneficial to plant chlorophyll synthesis, directly affecting plant photosynthesis and thus promoting plant growth. In conclusion, Amm-2 inoculation improves the quality of compost products and is beneficial to crop growth.

[0061] Finally, it should be noted that the above enumeration is only several specific embodiments of the present application. Obviously, the present application is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or inferred from the disclosure of the present application by those of ordinary skill in the art should be considered as falling within the scope of protection of the present application.

Claims

1. A fusiform Bacillus lysine (Bacillus fusiformis) characterized in that, Lysinibacillus fusiformis It is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20242776. ​ 2. The fermentation method of the spindling Bacillus lysiniformis according to claim 1, characterized in that, The method comprises the following steps: The fusiform lysinibacillus is inoculated into an ammonification culture medium and cultured at 32-37°C for 3-7 days.

3. A fermentation liquor obtained by the method according to claim 2.

4. An ammoniating bacterial inoculant characterized by, It is a product containing the fusiform lysinibacillus according to claim 1 or the fermentation liquor according to claim 3 as an active ingredient.

5. The ammonifying bacterial inoculant of claim 4, wherein, It is a product containing the fusiform lysinibacillus or the fermentation liquor and a microbiologically acceptable adjuvant.

6. Use of the fusiform lysinibacillus according to claim 1 or the fermentation liquor according to claim 3 or the ammonification bacterial agent according to claim 4 in composting.

7. Use according to claim 6, characterized in that, The ammonification bacterial agent or the fermentation liquor or the ammonification bacterial agent is used for promoting the ammonification process.

8. Use according to claim 7, characterized in that, The ammonification bacterial agent or the fermentation liquor or the ammonification bacterial agent is used for promoting the composting process of cow dung and improving the quality of the composting product.

9. A fertilizer, characterized in that, It is a product obtained by inoculating the fusiform lysinibacillus according to claim 1 or the fermentation liquor according to claim 3 or the ammonification bacterial agent according to claim 4 into composting materials for composting fermentation.

10. Use of the fertilizer according to claim 9 for promoting plant growth, characterized in that, The fertilizer is used for promoting the accumulation of chlorophyll and biomass in plants, or promoting the growth of plant height and root length.

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