Bacillus velezensis LU4 and application thereof

Abstract

The invention relates to bacillus velezensis LU4 and application thereof, the bacillus velezensis LU4 can promote straw degradation and enhance the cellulose degradation efficiency by inducing up-regulation expression of a cellulose degradation gene, and the bacillus velezensis LU4 shows good application potential in the aspect of straw resource utilization.

Description

Technical Field

[0001] This disclosure relates to the field of agricultural microbiology technology, specifically to a Bacillus belyssus LU4 and its applications. Background Technology

[0002] Crop straw components such as lignocellulose and silicates can form complex three-dimensional structures, making them difficult to degrade naturally. Directly returning straw to the field often breeds pathogenic microorganisms, leading to increased crop diseases and pests, severely impacting crop growth and reducing yields. Direct burning of straw produces large amounts of greenhouse gases, causing environmental pollution and hindering sustainable agricultural development. To efficiently utilize straw resources, long-term scientific research has led to increasingly diversified straw degradation pathways, mainly categorized into physical, chemical, and biological methods. Among these, biological (microbial) straw degradation offers advantages such as high degradation efficiency, significant energy-saving potential, cost-effectiveness, and environmental friendliness, making it a promising approach.

[0003] Efficient enzymatic hydrolysis of cellulose is a key technological bottleneck for the resource utilization of straw. Currently known cellulose-degrading bacteria are mainly concentrated in Trichoderma (…). Trichoderma ), Penicillium and other filamentous fungi ( Penicillium ), and Clostridium thermophilum ( Clostridium thermocellum ), cellulose-degrading heat-loving bacteria ( Acidothermuscellulolyticus Anaerobic bacteria such as Bacillus. Bacillus Due to its strong ability to secrete extracellular enzyme systems and its wide environmental adaptability, *Bacillus belyssioides* (BBE) has attracted much attention among aerobic degradative bacteria. Previous studies have shown that some *Bacillus belyssioides* (BBE)... Bacillus velezensis Bacillus belyssus can secrete key enzymes such as endoglucanase and exoglucanase, showing preliminary potential for cellulose degradation. However, existing strains generally suffer from low enzyme synergistic efficiency, poor overall enzyme activity, and significant product inhibition due to the lack of β-glucosidase, which limits their practical application. Therefore, breeding a new strain of Bacillus belyssus with a complete enzyme system, significant synergistic effects, and adaptability to complex field environments is of great significance for promoting in-situ straw return to the field and the high-value utilization of agricultural waste. Summary of the Invention

[0004] To address the technical problems in the prior art, this disclosure provides a strain of Bacillus belye LU4 and its applications. This strain can grow stably under various environmental conditions and exhibits significant cellulose degradation characteristics.

[0005] Firstly, this disclosure provides a Bacillus belesiensis (B. belesiensis) Bacillus velezensisLU4, this strain has been deposited at the China Center for Type Culture Collection (CCTCC) designated by the State Intellectual Property Office on June 15, 2023. The deposit number is CCTCC NO:M20231028.

[0006] In some embodiments, the Bacillus belales LU4 is isolated from humus soil.

[0007] In some embodiments, the 16S rDNA gene sequence of the Bacillus belyssus LU4 is shown in SEQ ID NO.1.

[0008] In some embodiments, the Bacillus berberis LU4 colonies on LB medium are round, opaque, smooth, and moist; under a microscope, Gram-stained strains appear blue-purple, indicating they are Gram-positive bacteria with short rod-shaped cells.

[0009] In some embodiments, the Bacillus belales LU4 can efficiently produce a variety of cellulase-degrading enzymes, such as filter paper enzyme, endoglucanase, exoglucanase, and β-glucosidase.

[0010] In some embodiments, the filter paper enzyme activity produced by *Bacillus belyssioides* LU4 is 109.82±0.794 U / mL, the endoglucanase activity is 80.13±0.60 U / mL, the exoglucanase activity is 54.91±2.42 U / mL, and the β-glucosidase activity is 67.36±0.45 U / mL.

[0011] In a second aspect, this disclosure provides a cellulose degrading agent, said cellulose degrading agent comprising Bacillus vesiculosus LU4 as described in the first aspect.

[0012] Thirdly, this disclosure provides the application of Bacillus berleis LU4 in the production of cellulase, wherein Bacillus berleis LU4 is the Bacillus berleis LU4 described in the first aspect.

[0013] In some embodiments, the cellulase includes filter paper enzymes, endoglucanases, exoglucanases, and β-glucosidases.

[0014] In some embodiments, the optimal pH for the cellulase activity produced by *Bacillus belyssiensis* LU4 is about 4.0-9.0, for example, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or about 9; the optimal temperature is about 40°C-70°C, for example, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, or about 70°C; and the optimal salt concentration is about 0 μM-2 μM, for example, about 0 μM, 0.5 μM, 1 μM, 1.5 μM, or 2 μM.

[0015] Fourthly, this disclosure provides a microbial compound fertilizer, comprising Bacillus vesiculus LU4 as described in the first aspect and excipients that are well compatible with Bacillus vesiculus LU4.

[0016] Based on the above technical solution, the embodiments of this disclosure can produce at least the following technical effects: (1) This disclosure is based on Bacillus belyss LU4, which was screened from low-temperature humus soil conditions. Bacillus velezensis This strain (LU4) exhibits stable growth and efficient production of cellulase, with filter paper enzyme activity of 109.82±0.794 U / mL, endoglucanase activity of 80.13±0.60 U / mL, exoglucanase activity of 54.91±2.42 U / mL, and β-glucosidase activity of 67.36±0.45 U / mL, demonstrating significant cellulose degradation characteristics. Furthermore, this strain exhibits strong environmental adaptability; the produced cellulase can effectively degrade cellulose under acidic or alkaline conditions, or in high-temperature or saline environments, thereby promoting straw degradation. It further enhances cellulose degradation efficiency by inducing the upregulation of cellulose degradation genes, showing good application potential in the resource utilization of straw.

[0017] (2) The present invention discloses a cellulose degrading agent prepared from Bacillus belye LU4. Under the condition of using 3% sodium carboxymethyl cellulose as the sole carbon source, Bacillus belye LU4 significantly outperformed its performance in cellulose degradation and induction of cellulose degradation gene expression in LB medium. This demonstrates that the strain can effectively respond to cellulose substrates and initiate the degradation process, providing a basis for the development of highly efficient cellulose-degrading microbial agents. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0019] Figure 1 The colony morphology and Gram staining of Bacillus belyss LU4 are shown. Figure 2 Cellulase activity of Bacillus belyssus LU4; Figure 3 The activity of Bacillus belysin LU4 enzyme in culture media with different pH, temperature and salt concentrations was measured. Figure 4 Physiological and biochemical identification of Bacillus belyss LU4; Figure 5 Phylogenetic tree of Bacillus belyss LU4 based on 16S rDNA gene sequence; Figure 6 A schematic diagram of the complete genome structure of Bacillus belyssus LU4; Figure 7 This is based on annotation results from the NR database; Figure 8 Phylogenetic tree diagram of LU4 based on Bacillus belysium; Figure 9 This is a diagram showing the comparative analysis of closely related strains based on ANI analysis; Figure 10 Annotated figure for Bacillus belyssus LU4 based on COG, KEGG, and Cazymes databases; Figure 11 This is a diagram showing the expression analysis of the LU4 gene in Bacillus belysaeus based on RT-qPcr. Figure 12 This is a graph showing the enrichment and function of cellulose degradation products. Figure 13 This is a diagram illustrating the straw degradation process based on solid-state fermentation. Figure 14 This is a graph showing the change in nitrogen content based on solid-state fermentation alkaline hydrolysis. Figure 15 This is a graph showing the change in available phosphorus content based on solid-state fermentation. Figure 16 This is a graph showing the change in available potassium content based on solid-state fermentation. Figure 17 This is a graph showing the change in organic matter content based on solid-state fermentation; Figure 18 This is a graph showing the change in neutral detergent fiber content based on solid-state fermentation; Figure 19 This is a graph showing the change in fiber content in acidic detergents based on solid-state fermentation. Detailed Implementation

[0020] The technical solutions in the embodiments of this disclosure will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. Based on the embodiments in this disclosure, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this disclosure. In addition, the technical solutions of various embodiments can be combined with each other, but this must be based on the ability of those of ordinary skill in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by this disclosure.

[0021] Unless otherwise specified, all reagents used in this disclosure are commercially available reagents; unless otherwise specified, all techniques and experimental methods used in this disclosure are conventional techniques and methods in the field.

[0022] Example 1: Isolation and screening of Bacillus belye LU4 To obtain strains capable of effectively degrading cellulose, strains were isolated and purified from humic soil using the streak plate method. The purified strains were then acclimatized in LB medium, and subsequently inoculated onto CMC, Avicel, and starch media for cultivation. The cellulose degradation capacity was analyzed by measuring the size of the degradation zone. In CMC medium... Bacillus velezensis The degradation zone of LU4 is the largest, indicating that Bacillus belyeis LU4 has a strong cellulose degradation ability (Table 1).

[0023] Table 1. Determination of hydrolysis ratio and degradation zone diameter of isolated strains on CMC medium.

[0024] Example 2: Analysis of the physiological and biochemical characteristics of Bacillus belyssus LU4 1. Morphology of Bacillus belyss LU4 Bacillus berberis LU4 ( Bacillus velezensis LU4) was cultured on LB medium under the following conditions: 37℃, 150 rpm, 24 h; the results showed that the strain grew well, as shown in the attached... Figure 1 As shown, the colonies are round, opaque, smooth, and moist. Under a 10×100 microscope, the Gram-stained strain appears blue-purple, indicating it is a Gram-positive bacterium with short rod-shaped cells.

[0025] 2. Determination of LU4 enzyme activity and enzyme kinetics in Bacillus belyssus 2.1 Determination of enzyme activity in cellulose-degrading bacteria The purified strain was inoculated into LB liquid medium and activated at 37°C, 150 rpm for 24 h. Then, the bacterial culture was inoculated into an Erlenmeyer flask containing 100 mL of LB medium and fermented at 37°C, 170 rpm for 1 day. After fermentation, the bacterial culture was aspirated and centrifuged at 12000 rpm, 4°C for 10 min. The supernatant was then collected as the crude enzyme solution.

[0026] (1) Determination of total cellulase activity The enzyme activity was determined using the filter paper enzyme activity (FPA) method. 0.1 g of filter paper strip was added to a stoppered colorimetric tube, followed by 2 mL of citrate-disodium hydrogen phosphate buffer (pH 4.8) to moisten the filter paper strip. 1 mL of crude enzyme solution was added and vortexed to mix (1 mL of sterile crude enzyme solution was used for the blank control). The tube was incubated at 50 °C for 30 min, then 3 mL of DNS reagent was added. After mixing, the tube was boiled in a water bath for 10 min, cooled to room temperature, and brought to a final volume of 20 mL. The absorbance was measured at 540 nm. Each tube was repeated three times.

[0027] (2) Determination of endo-β-1,4-glucosidase activity The activity of carboxymethyl cellulose (CMC) enzyme was determined using a method that involves adding 2 mL of 1% CMC-Na solution (prepared with citrate-disodium hydrogen phosphate buffer at pH 4.8) to a stoppered colorimetric tube, followed by the addition of 1 mL of crude enzyme solution and vortexing (the blank group consisted of 1 mL of sterile crude enzyme solution). The tube was incubated at 50 °C for 30 min, then 3 mL of DNS reagent was added. After mixing, the tube was boiled in a water bath for 10 min, cooled to room temperature, and brought to a final volume of 20 mL. The absorbance was measured at 540 nm. Each tube was repeated three times.

[0028] (3) Determination of exo-β-1,4-glucosidase activity The microcrystalline cellulose (MCC) enzyme activity assay was performed. 2 mL of 1% microcrystalline cellulose solution (prepared with citrate-disodium hydrogen phosphate buffer at pH 4.8) was added to a stoppered colorimetric tube. 1 mL of crude enzyme solution was added and vortexed to mix (the blank group consisted of 1 mL of sterile crude enzyme solution). The tube was incubated at 50°C for 30 min. 3 mL of DNS reagent was added, and the mixture was boiled in a water bath for 10 min. After cooling to room temperature, the volume was adjusted to 20 mL. The absorbance was measured at 540 nm. Each tube was repeated three times.

[0029] (4) β-1,4-glucosidase activity assay The salicylase activity assay was performed. 2 mL of 1% salicin solution (prepared with citrate-disodium hydrogen phosphate buffer at pH 4.8) was added to a stoppered colorimetric tube. 1 mL of crude enzyme solution was added and vortexed to mix (1 mL of sterile crude enzyme solution was used for the blank control). The tube was incubated at 50°C for 30 min. 3 mL of DNS reagent was added, and the mixture was boiled in a water bath for 10 min. After cooling to room temperature, the volume was adjusted to 20 mL. The absorbance was measured at 540 nm. Each tube was repeated three times.

[0030] (5) Cellulase activity calculation: One unit (U) of enzyme activity is defined as the amount of enzyme required to release 1 µmol of reducing sugar per minute per milliliter. The formula for calculating cellulase activity is as follows: Enzyme activity (U / mL) = Glucose content (mg) × 1000 / Crude enzyme solution volume (mL) × Reaction time (min).

[0031] Enzyme activity assay results as follows Figure 2 As shown, the filter paper enzyme activity of Bacillus belyss LU4 was 109.82±0.794 U / mL, the endoglucanase activity was 80.13±0.60 U / mL, the exoglucanase activity was 54.91±2.42 U / mL, and the β-glucosidase activity was 67.36±0.45 U / mL. All four enzymes showed high activity, indicating that Bacillus belyss LU4 can effectively degrade cellulose.

[0032] 2.2 Optimal Enzyme Activity Conditions for Determining Cellulose-Degrading Enzymes (1) To determine the optimal pH value for the cellulase activity produced by LU4, we also conducted a gradient experiment and prepared citrate-disodium hydrogen phosphate buffer solutions with different pH values ​​(pH 3~9). The enzyme was added to the reaction mixture and reacted at 50°C. Three parallel experimental groups were set up for each pH gradient, and the activity was then determined based on the 3,5-dinitrosalicylic acid (DNS) colorimetric method.

[0033] The results are as follows Figure 3 As shown in a, all four enzymes maintained high enzyme activity within the pH range of 4-9, with the highest overall enzyme activity, especially within the pH range of 4-6.

[0034] (2) To determine the optimal enzyme activity temperature of the cellulase produced by LU4, the enzyme solution and substrate were incubated in pH buffer (pH 5) at different temperatures (10~80 ℃) for 30 min. Three parallel experimental groups were set up for each temperature gradient. Then, the optimal reaction temperature of LU4 was determined based on the DNS method.

[0035] The results are as follows Figure 3As shown in b, the cellulose-degrading enzyme produced by LU4 has a wide range of suitable enzyme activity temperatures, with high enzyme activity in the range of 20-80℃, especially under medium temperature (40-70℃) conditions, the overall enzyme activity is the highest.

[0036] (3) To analyze the effect of salt concentration on the enzyme activity of cellulase, sodium chloride solutions of different concentrations were added to the experimental reaction system, and the reaction was carried out at 40℃ and pH 6 for 30 min. The enzyme reaction system without added salt solution was used as the control group to determine the effect of salt concentration on enzyme activity.

[0037] The results are as follows Figure 3 As shown in c, the cellulase produced by LU4 is highly adaptable to the salt environment and has high enzyme activity in the 0-4 μM salt environment, especially under the low salt 0-2 μM condition, the overall enzyme activity is the highest.

[0038] In summary, the four cellulase systems produced by Bacillus belyss LU4 exhibited superior overall enzyme activity under weakly acidic (pH 4-6), mesophilic (40-70℃), and low-salt (0-2μM) conditions. Furthermore, the different enzymes showed specific responses to environmental factors, demonstrating the functional diversity and environmental adaptability of the enzymes produced by Bacillus belyss LU4.

[0039] 3. Identification of the physiological and biochemical characteristics of the strain The metabolic characteristics of the strain were identified using the BIOLOG GeneⅢ system. Results are as follows: Figure 4 As shown, strain LU4 can utilize 31 carbon source substrates, including dextrin, maltose, trehalose, cellobiose, sucrose, D-fructose, and D-mannose, for growth. Among them, 11 sugar alcohol substrates showed positive reactions, including α-D-glucose, D-mannose, and D-fructose (Table 2). It also showed positive reactions with 4 amino acid substrates, indicating that LU4 can utilize L-alanine, L-aspartic acid, L-glutamic acid, and D-aspartic acid substrates for growth (Table 3). It showed positive reactions with 5 antibiotic substrates, including sodium butyrate, sodium sulfite, lithium chloride, sodium lactate, and guanidine hydrochloride, while being sensitive to antibiotics such as rifamycin SV and dimethylaminotetracycline, indicating that strain LU4 does not have tolerance to some antibiotics (Table 4).

[0040] Table 2. Identification of Bacillus belyss LU4 using sugar alcohol reactions in GenIII identification plates.

[0041] Table 3. Amino acid reaction identification of Bacillus belyss LU4 in GenIII identification plates.

[0042] Table 4. Antibiotic reaction identification of Bacillus belyss LU4 in GenIII identification plates

[0043] Example 3: Molecular biological identification of Bacillus belyss LU4 1. Molecular biological identification based on 16S rDNA The PCR amplification product of 16S rDNA from strain LU4 was sequenced, and the sequence number is shown in SEQ ID NO. 1. SEQ ID NO. 1: In the NCBI database, homology searches were performed using Blester and published 16S rDNA sequences in GenBank, and similarity comparisons were used to identify strain LU4 as similar to... Bacillus The 16S rDNA sequence alignment of sp. showed the highest consistency, with a similarity exceeding 99%. Therefore, the 16S rDNA gene sequences of relevant strains were downloaded from GeneBank, and multiple comparisons were performed using MEGA 11.0 software to construct a phylogenetic tree. The similarity was then determined based on genetic distance. LU4 and... Bacillus velezensis strain FZB42 、 The similarity is 100% ( Figure 5 Therefore, LU4 belongs to... Bacillus .

[0044] 2. Whole genome analysis Whole-genome sequencing revealed that strain LU4 exhibited a circular chromosome structure, with a genome size of 3,929,792 bp and a GC content of 46.5%. Functional annotation yielded one prophage and one gene island, annotating 3,747 coding genes, including 27 rRNA genes and 86 tRNA genes. Figure 6 ), 3669 genes were obtained from the Nr database comparison, LU4 and Bacillus velezensis The maximum number of genes shared is 2808, and LU4 may belong to [a specific gene group]. Bacillus velezensis ( Figure 7 ).

[0045] 3. Comparative genomic analysis with other Bacillus species, including Belize. The phylogenetic tree constructed based on 211 Bacillus belyeis and LU4 whole genome sequences showed that strain LU4 is related to... Bacillus velezensis ZLP-10 (CP128992.1) is most closely related to ( Figure 8 OrthoANI values ​​of the genome sequences of LU4 and six Bacillus species showed that strain LU4 was related to... Bacillus velezensis The similarity of IFST-221 (CP125283.1) reached 98.77%, followed by... Bacillus velezensis strain FZB42 (NC_009725) (98.27%) Bacillus velezensis strain SSF6(CP12945) (97.77%) Bacillus velezensis strain CL-4(CP081304.1)(97.73%) Figure 9 In conclusion, strain LU4 has been confirmed as Bacillus belye, and has been named [name missing]. Bacillus velezensis LU4.

[0046] Example 4: Genomic structural characteristics of Bacillus belyssus LU4 1. Annotation analysis based on COG database COG database annotations are categorized into 23 functional types. Figure 10 a) The top three functional types enriched by genes were Amino acid transport and metabolism (307 genes), Transcription (294 genes), and Carbohydrate transport and metabolism (278 genes), accounting for a total of 28.6% of the annotated genes, indicating that the genome of Bacillus belyss LU4 contains abundant nutritional metabolism genes.

[0047] 2. Based on carbohydrate metabolic pathways closely associated with cellulose degradation in the KEGG database The KEGG database identified 15 carbohydrate metabolism pathways closely associated with cellulose degradation, involving 425 genes. Figure 10 b). Pyruvate metabolism (ko00620, 52 genes), Amino sugar and nucleotide sugar metabolism (ko00520, 47 genes), Starch and sucrose metabolism (ko00500, 46 genes), and glycolysis / gluconeogenesis (ko00010, 41 genes) are dominant pathways and play a key role in energy metabolism during cellulose degradation. These results indicate that the Bacillus bereaves LU4 genome contains abundant genes related to carbohydrate and other metabolism.

[0048] 3. Annotation analysis based on CAZy database Annotation analysis of the CAZy database revealed that a total of 128 CAZyme genes were identified in the genome. Figure 10 c), mainly distributed in five major functional groups: glycosyltransferases (GT, 42), glycosidases (GH, 41), glycoesterases (CE, 31), cofactor enzymes (AA, 9), and polysaccharide lyases (PL, 3). Figure 12Seven genes are involved in cellulose degradation, two endocellulase genes encode GH51 family proteins, and five glucanase genes belong to the GH1 and GH3 families. Eleven genes are involved in hemicellulose degradation, such as β-xylosidase (GH1 / GH3 / GH51 family), xylanase (GH26 family), and mannanase (GH11 / GH51 family). Three hemicellulose degradation coenzyme genes, including α-L-arabinofuranylase, were also annotated. Lignin degradation-related genes include laccase (AA1) and polysaccharide monooxygenase (AA10). The abundance of cellulase genes indicates that LU4 has a strong cellulose degradation potential (Table 5).

[0049] Table 5. Annotated sequences encoding cellulose and lignin-degrading enzymes in Bacillus belyssus LU4

[0050]

[0051] Example 5: Upregulation of cellulose degradation-related genes in Bacillus belye LU4 under cellulose conditions Will Bacillus velezensis Culture medium of LU4 strain (concentration of 1×10⁻⁶) 8 CFU·mL -1 The strain was incubated at 28°C for 4 days under both CMC-Na and CMC-Na-free (CK) conditions, with enzyme activity monitored periodically. Results showed that *Bacillus belyssiensis* LU4 exhibited the highest enzyme activity at 72 hours. Relative gene expression levels were analyzed at 72 hours, and qRT-PCR results showed that compared to the control group (without CMC-Na), Bacillus velezensis LU4 bacterial inoculation significantly upregulated genes involved in cellulose degradation. CotA , nagZ, celB, xynB and abfA Relative expression level ( P <0.05)( Figure 11 The characteristics of their expression profiles are highly consistent with the previous transcriptome sequencing results, confirming the core role of these enzymes in the cellulose degradation process.

[0052] Example 6: Enrichment and Function of Cellulose Degradation Products from Bacillus belye LU4 under Cellulose Conditions Untargeted metabolomics analysis was performed on Bacillus belyssioides LU4 under CMC-Na-containing and CMC-Na-free (CK) conditions. Volcano plot results showed that 1188 differentially expressed metabolites (DAMs) were identified in the CMC-Na group, of which 823 were upregulated and 365 were downregulated. Figure 12a). KEGG enrichment analysis showed that upregulated metabolites were significantly enriched in 12 pathways, including histidine metabolism, valine / leucine / isoleucine degradation, and pyrimidine metabolism (P<0.01), involving a total of 65 key metabolites, including L-valine, nicotinamide, and UDP, of which 17 were related to carbohydrate metabolism. Downregulated metabolites were mainly enriched in 5 pathways, including nucleotide metabolism and neomycin / kanamycin / gentamicin biosynthesis. Figure 12 b). The CMC-Na group showed high expression levels of 15 metabolites, including carbohydrate derivatives, amino acids, and their derivatives. Multivariate statistical analysis (VIP≥1) combined with significance testing (…) P <0.05%, a total of 30 significantly differentially expressed metabolites were screened, of which 22 were upregulated and 8 were downregulated, mainly distributed in 7 classes of compounds including lipids, organic acids, and phenylpropanoids. Figure 12 c). This metabolomic profile reflects the systemic metabolic reprogramming characteristics of Bacillus belyss LU4 under cellulose induction.

[0053] Example 7: Determination of the effect of Bacillus vesiculosus LU4 on straw degradation To verify the effect of Bacillus vesiculosus LU4 on straw degradation, the following solid-state fermentation experiment was designed.

[0054] 1. Experimental Materials Experimental group: The purified strain was inoculated into LB liquid medium and activated by culturing at 37℃ and 150 rpm for 24 h, preparing a concentration of 1×10⁻⁶. 8 CFU·mL -1 Bacillus berberis LU4 bacterial culture; Control group: Sterile water (SW).

[0055] 2. Experimental Methods Set up two treatment groups, T1 and T2, with three replicates in each group: T1: Sterile water (SW) treatment (control); T2: Treatment with Bacillus belyss LU4 bacterial culture (1×10⁻⁶) 8 CFU·mL -1 ); The collected corn stalks were air-dried and then crushed into 5cm lengths. 20g of each stalk was weighed and placed into glass bottles, and the humidity was adjusted to 65%. The bottles were then sterilized at 121℃ for 30 min. Under aseptic conditions, 7 mL of each treatment group was added to the glass bottles, and the mixture was statically fermented at 25℃ for 35 days in an artificial incubator with a humidity of 75%.

[0056] 3. Measurement indicators: Changes in straw morphology, readily available nutrients in straw (available nitrogen, available phosphorus, available potassium), organic matter, acid detergent fiber, and neutral detergent fiber.

[0057] Electron microscopy observations of straw degradation experiments showed that, compared to the control group, the crude cellulase group of strain LU4 exhibited significant changes in the surface texture of the straw, including porosity, damage, and cavities. Figure 13 This indicates that the strain has a strong ability to degrade straw.

[0058] The content of alkaline nitrogen first decreased and then increased with the extension of fermentation time, showing an overall trend of "low-decline-increase". That is, in the early stage, Bacillus belales LU4 used nitrogen sources to multiply, which led to a decrease in alkaline nitrogen, and in the later stage, the release of nitrogen mineralization from straw caused alkaline nitrogen to rebound. Figure 14 ).

[0059] The content of available phosphorus shows a "continuous increase" trend with the extension of fermentation time, that is, the phytase secreted by Bacillus belye LU4 continuously decomposes the insoluble bound phosphorus (such as phytate phosphorus and calcium phosphorus) in straw, and continuously releases absorbable available phosphorus, and the content in the mature stage (35 days) needs to be significantly higher than that in the initial stage. Figure 15 ).

[0060] The content of available potassium showed a trend of "rapid increase followed by stabilization" with the extension of fermentation time. That is, in the early stage, Bacillus belales LU4 rapidly released bound potassium by destroying the straw cell wall through cellulase and hemicellulase; in the later stage, the cell wall structure was basically decomposed, the potassium release rate slowed down, and the content tended to stabilize, with no decline stage throughout the process. Figure 16 ).

[0061] The organic matter content remained "basically stable with slight fluctuations" as fermentation time increased. This means that *Bacillus belyceraea* LU4 primarily converted recalcitrant organic matter (such as crude fiber and lignin) in straw into easily absorbed active organic matter (such as humic acid), with no significant decrease in total organic matter content. Only slight fluctuations (≤5%) occurred due to the release of a small amount of organic carbon as CO2. Figure 17 ).

[0062] The content of neutral detergent fiber (NDF) showed a "continuous decline" trend with the extension of fermentation time. That is, the cellulase and hemicellulase secreted by Bacillus belye LU4 continuously degraded NDF (containing cellulose, hemicellulose, and lignin). Among them, cellulose and hemicellulose were efficiently decomposed, resulting in a gradual decrease in NDF content from the initial stage. The content at the mature stage (35 days) was significantly lower than that at the initial stage, and the decline process did not rebound. Figure 18 ).

[0063] The content of acid detergent fiber (ADF) showed a trend of "slow decrease and overall stability" with the extension of fermentation time. That is, ADF mainly contains cellulose and lignin. Bacillus belye LU4 can degrade cellulose but has difficulty decomposing lignin, resulting in a slight decrease in ADF in the early stage as cellulose degrades, and a stabilization in the later stage due to the relatively increased proportion of lignin. There were no significant fluctuations or increases throughout the process. Figure 19 ).

[0064] In summary, the lignocellulose degrading agent containing Bacillus belyss LU4 exhibits highly efficient and stable straw degradation performance, bringing significant and diverse beneficial effects. From a resource conversion perspective, this degrading agent, through the functional enzyme system such as crude cellulase and hemicellulase secreted by the strain, efficiently destroys the dense cell wall structure of straw, creating numerous pores and damage on the straw surface. This accelerates the decomposition and transformation of recalcitrant organic matter, converting previously unusable crude fiber and lignin in straw into readily absorbable active organic matter such as humic acid. This achieves both the harmless treatment of straw resources and the conversion of agricultural waste into high-quality organic nutrients, improving resource recycling rates. Regarding nutrient supply, the degradation process continuously releases bound phosphorus and potassium elements from the straw, causing a sustained increase in available phosphorus content and a rapid increase in available potassium content in the fermentation system, which then stabilizes at a high level. Simultaneously, through the synergistic effect of strain growth and reproduction and straw nitrogen mineralization, a dynamic balance of alkaline nitrogen content is maintained, ultimately providing balanced and sufficient available nitrogen, phosphorus, and potassium nutrients for crop growth, effectively compensating for the problem of insufficient available nutrients in the soil. From a soil improvement perspective, the stable total amount of organic matter retained after degradation and the humic acid generated help improve soil aggregate structure, enhance soil water and fertilizer retention capacity, and improve soil aeration. The continuous decrease in neutral detergent fiber (NDF) and the stable control of acid detergent fiber (ADF) prevent soil compaction caused by the accumulation of undegraded fibers. Simultaneously, the combined action of bacterial metabolic activity and straw degradation products optimizes the soil micro-ecological environment, creating favorable conditions for crop root growth. Furthermore, the application of this degrading agent can reduce environmental pollution caused by straw burning or indiscriminate dumping, reduce reliance on chemical fertilizers, and has significant practical value in improving crop yield and quality and promoting sustainable agricultural development. It provides an efficient and feasible technical solution for the resource utilization of straw and the construction of ecological agriculture.

[0065] The foregoing has shown and described the basic principles, main features, and advantages of this disclosure. Those skilled in the art should understand that this disclosure is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of this disclosure. Various changes and modifications can be made to this disclosure without departing from its spirit and scope, and all such changes and modifications fall within the scope of this disclosure as claimed. The scope of protection of this disclosure is defined by the appended claims and their equivalents.

Claims

1. A type of Bacillus belesii ( Bacillus velezensis LU4, characterized in that, The Bacillus belyss LU4 was deposited on June 15, 2023, by the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M20231028.

2. The Bacillus belyssus LU4 according to claim 1, characterized in that, The Bacillus berberis LU4 was isolated from humus soil.

3. The Bacillus belyssus LU4 according to claim 1, characterized in that, The 16S rDNA gene sequence of the Bacillus vesiculosus LU4 is shown in SEQ ID NO.

1.

4. The Bacillus belyssus LU4 according to claim 1, characterized in that, The Bacillus berberis LU4 colonies on LB medium are round, opaque, smooth, and moist. Under a microscope, the Gram-stained strain appears blue-purple, indicating it is a Gram-positive bacterium with short rod-shaped cells.

5. The Bacillus belyssus LU4 according to claim 1, characterized in that, The filter paper enzyme activity produced by *Bacillus belyssioides* LU4 was 109.82±0.794 U / mL, the endoglucanase activity was 80.13±0.60 U / mL, the exoglucanase activity was 54.91±2.42 U / mL, and the β-glucosidase activity was 67.36±0.45 U / mL.

6. A cellulose degrading agent, characterized in that, Includes Bacillus berberis LU4 as described in any one of claims 1 to 5.

7. The application of Bacillus belales LU4 according to claims 1 to 5 in the production of cellulase, preferably, the cellulase includes filter paper enzyme, endoglucanase, exoglucanase and β-glucosidase.

8. The application according to claim 7, characterized in that, The optimal pH for the cellulase produced by *Bacillus belyssus* LU4 is approximately 4.0-9.0, for example, approximately 4.0, approximately 4.5, approximately 5.0, approximately 5.5, approximately 6.0, approximately 6.5, approximately 7.0, approximately 7.5, approximately 8.0, approximately 8.5, and approximately 9. The optimal temperature is approximately 40 ℃-70 ℃, for example, approximately 40 ℃, approximately 45 ℃, approximately 50 ℃, approximately 55 ℃, approximately 60 ℃, approximately 65 ℃, and approximately 70 ℃. The optimal salt concentration is approximately 0 μM-2 μM, for example, approximately 0 μM, 0.5 μM, 1 μM, 1.5 μM, and 2 μM.

9. The application of Bacillus belales LU4 according to any one of claims 1 to 5 or the cellulose degrading agent according to claim 6 in the degradation of straw.

10. A microbial compound fertilizer, characterized in that, It includes Bacillus berberis LU4 as described in claims 1 to 5 and excipients that are compatible with Bacillus berberis LU4.

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