A Bacillus hainanensis with the function of degrading lignocellulose and its application

Abstract

The present invention provides a new species of Bacillus, named Bacillus hainanensis XZB-5 (Bacillus hainanensis), which is registered and preserved in the Guangdong Provincial Microbial Culture Collection Center with the preservation number GDMCC NO: 64531. The Bacillus hainanensis of the present invention has good enzyme activities such as endoglucanase, exoglucanase, and β-glucosidase, and can effectively degrade cellulose and hemicellulose. It also has good enzyme activities such as manganese peroxidase, lignin peroxidase, and laccase, and can effectively degrade lignin. It has a high-efficiency degradation function for agricultural residues and the like, can rapidly decompose agricultural residues and the like, release nutrients, provide nutrition for other microorganisms in the environment, improve the soil microecology, avoid environmental pollution, and has high practical value in the resource utilization of waste.

Description

Technical Field

[0001] The present invention relates to the biological field, and specifically relates to a Bacillus hainanensis with the function of degrading lignocellulose and its application. Background Art

[0002] With the increasing global concern for environmental sustainability, the effective treatment and utilization of agricultural and forestry waste have become key issues. Especially in forestry activities, a large amount of bark produced as a by-product has a direct impact on environmental protection and resource recovery efficiency (Wang et al., 2022). As a rich source of lignocellulose, bark decomposes slowly under natural conditions, not only occupying a large amount of land space but also potentially becoming a breeding ground for pests, thus challenging the sustainable development of forestry. Therefore, exploring efficient bark composting technologies to accelerate its conversion into high-quality organic fertilizers is the key solution to this challenge (Chen et al., 2021).

[0003] Composting is a process of stabilizing and detoxifying organic waste, which not only reduces the negative environmental impact of waste but also converts it into organic fertilizers beneficial to agricultural production (Qu et al., 2023). However, the high content of lignocellulose in bark makes it difficult to decompose rapidly under natural conditions, limiting the efficiency of the composting process and the quality of the final product (Jia et al., 2023). Therefore, it is crucial to discover an appropriate strategy that can accelerate the degradation of lignocellulose during composting. In this context, adding exogenous lignocellulose-degrading microorganisms has become an effective strategy to improve the efficiency of bark composting (Nigussie et al., 2021). These microorganisms accelerate the decomposition of lignocellulose by secreting a series of enzymes, including cellulase and ligninase, thus promoting the rapid conversion and maturation of organic matter during composting (Peng et al., 2021). Therefore, screening microorganisms capable of efficiently degrading lignocellulose is a prerequisite for lignocellulose biodegradation.

[0004] In recent years, some fungi, including Phanerochaete ( Phanerochaete ), Trichoderma ( Trichoderma ), Trametes ( Trametes ), and Aspergillus ( Aspergillus ), have been widely used in composting experiments due to their significant lignin degradation ability to improve fermentation performance (He et al., 2022; Sajid et al., 2022). However, due to the sensitivity of fungi to the surrounding environment and temperature, and the fact that some of them may produce secondary metabolites harmful to plant growth, it limits their application in compost products. More and more researchers are focusing on isolating bacteria with the ability to degrade lignocellulosic biomass, such as Bacillus (Bacillus ), Fictibacillus , Acetobacter( Acetobacter ) and Aneurinibacillus (Chen et al., 2021; Chen et al., 2020; Wang et al., 2022; Wu et al., 2022). Among them, species of the genus Bacillus have shown significant ability to degrade cellulose-rich agricultural and forestry by-products. Their addition has been proven to promote the rapid conversion and maturation of organic matter during composting (Wu et al., 2022). Previous studies have shown that inoculating cellulose-degrading microorganisms can affect the microbial community structure (Wu et al., 2022), accelerate compost maturation (Yu et al., 2020), enhance the conversion of organic matter (Sajid et al., 2022), increase the activity of lignocellulolytic enzymes (Chen et al., 2021) and accelerate the humification process (Jia et al., 2023). However, there is still a lack of strains that can efficiently degrade lignocellulose. Therefore, the efficient utilization of cellulose is an important direction in the research of waste resource utilization. SUMMARY OF THE INVENTION

[0005] The object of the present invention is to overcome the deficiencies in the prior art and provide a new species of Bacillus with the function of degrading lignocellulose, namely Bacillus hainanensis, and also provide the application of this new species of Bacillus hainanensis.

[0006] The first aspect of the present invention is to provide a Bacillus hainanensis, which is a new species of Bacillus, named Bacillus hainanensis XZB-5, deposited in the Guangdong Provincial Culture Collection of Microorganisms, with the deposit number GDMCC NO: 64531.

[0007] The second aspect of the present invention is to provide the fermentation broth of the Bacillus hainanensis described in the first aspect of the present invention.

[0008] The third aspect of the present invention is to provide a microbial agent containing the Bacillus hainanensis described in the first aspect of the present invention.

[0009] The fourth aspect of the present invention is to provide a lignin-degrading enzyme preparation containing the fermentation broth or the sterile filtrate of the fermentation broth of the Bacillus hainanensis described in the first aspect of the present invention.

[0010] The fifth aspect of the present invention is to provide the application of the Bacillus hainanensis described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the microbial agent described in the third aspect of the present invention, or the lignin-degrading enzyme preparation described in the fourth aspect of the present invention in degrading cellulose, and / or hemicellulose, and / or lignin.

[0011] The sixth aspect of the present invention is to provide the application of Bacillus hainanensis described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the bacterial agent described in the third aspect of the present invention, or the lignin-degrading enzyme preparation described in the fourth aspect of the present invention in the preparation of a preparation producing exoglucanase, and / or endoglucanase, and / or β-glucosidase, and / or manganese peroxidase, and / or lignin peroxidase, and / or laccase.

[0012] The seventh aspect of the present invention is to provide the application of Bacillus hainanensis described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the bacterial agent described in the third aspect of the present invention, or the lignin-degrading enzyme preparation described in the fourth aspect of the present invention in the degradation of agricultural residues.

[0013] The eighth aspect of the present invention is to provide the application of Bacillus hainanensis described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the bacterial agent described in the third aspect of the present invention, or the lignin-degrading enzyme preparation described in the fourth aspect of the present invention in the degradation of eucalyptus bark, and / or peanut shells, and / or rice straw, and / or soybean meal, and / or corn bran, and / or rice husks, and / or rapeseed straw, and / or wheat straw, and / or banana straw.

[0014] The ninth aspect of the present invention is to provide the application of Bacillus hainanensis described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the bacterial agent described in the third aspect of the present invention, or the lignin-degrading enzyme preparation described in the fourth aspect of the present invention in increasing the seed germination rate.

[0015] The tenth aspect of the present invention is to provide the application of Bacillus hainanensis described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the bacterial agent described in the third aspect of the present invention, or the lignin-degrading enzyme preparation described in the fourth aspect of the present invention in the preparation of fertilizers.

[0016] The said Bacillus hainanensis can accelerate the transformation of compost products and shorten the composting period.

[0017] The eleventh aspect of the present invention is to provide a fertilizer containing Bacillus hainanensis described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the bacterial agent described in the third aspect of the present invention, or the lignin-degrading enzyme preparation described in the fourth aspect of the present invention.

[0018] The Bacillus hainanensis of the present invention is a new species of Bacillus, which has good enzyme activities such as endoglucanase, exoglucanase, and β-glucosidase, can effectively degrade cellulose and hemicellulose, and also has good enzyme activities such as manganese peroxidase, lignin peroxidase, and laccase, can effectively degrade lignin, has a high-efficiency degradation function for agricultural residues, etc., can rapidly decompose agricultural residues, release nutrients, provide nutrition for other microorganisms in the environment, improve the soil microecology, avoid environmental pollution, and has high practical value in the resource utilization of waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the phylogenetic tree of strain XZB-5.

[0020] Figure 2 It is the circular genome map of strain XZB-5 and the comparison results of ANI and DDH values with the reference strain.

[0021] Figure 3 It is the detection results of cellulase and ligninase activities produced by strain XZB-5.

[0022] Figure 4 It is the detection results of compost temperature, conductivity, and seed germination rate of strain XZB-5 in bark compost.

[0023] Figure 5 It is the detection results of cellulase and ligninase activities produced by strain XZB-5 in bark compost. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments for better understanding. For those not specified in the embodiments, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0025] The present invention provides a Bacillus hainanensis, which is a new species of Bacillus, named Bacillus hainanensis ( Bacillus hainanensis ) XZB-5, registered and preserved in the Guangdong Provincial Culture Collection of Microorganisms, with the preservation number GDMCC NO: 64531, the preservation date being April 22, 2024, and the preservation address being the 5th floor of Building 59, No. 100, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. The Bacillus hainanensis XZB-5 of the present invention was isolated and screened from the stony coral of Wuzhizhou Island, Hainan.

[0026] 1 Experimental Materials

[0027] 1.1 Sample Collection

[0028] Three coral samples were collected from waters at a depth of approximately 15 - 20 meters on Wuzhizhou Island (18°18′51"N, 109°45′42"E), Hainan Province, China. The three coral samples were placed into a sterile sealable bag, mixed evenly, sealed, numbered, stored in an ice box, and then sent to the laboratory for the isolation of co - associated bacteria.

[0029] 1.2 Main reagents

[0030] (1)DNS reagent: Weigh 10 g of 3,5 - dinitrosalicylic acid and dissolve it in 500 mL of distilled water. Add 20 g of sodium hydroxide and 200 g of potassium sodium tartrate. Heat the solution until it dissolves, then add 2 g of phenol and 0.5 g of anhydrous sodium sulfite. After the solutes dissolve, cool the solution to room temperature and make up the volume to 1000 mL. The reagent is stored in a brown volumetric flask and filtered for use after standing for one week (Wang Lin, 1999).

[0031] (2)Sodium acetate buffer solution: Weigh 16.4 g of sodium acetate and dissolve it in 1000 mL of distilled water to form a 0.2 mol / L buffer solution with a pH of 4.6.

[0032] (3)CMC substrate solution: Weigh 0.625 g of sodium carboxymethyl cellulose and dissolve it in 100 mL of sodium acetate buffer solution by heating and stirring until dissolved.

[0033] (4)Microcrystalline cellulose substrate solution: Weigh 0.5 g of microcrystalline cellulose and dissolve it in 100 mL of sodium acetate buffer solution.

[0034] (5)Filter paper substrate solution: Weigh 0.5 g of filter paper and immerse it in 100 mL of sodium acetate buffer solution.

[0035] (6)Sodium hydroxide solution: Weigh 4 g of sodium hydroxide and dissolve it in 50 mL of distilled water to prepare a 2 mol / L sodium hydroxide solution.

[0036] (7)Standard glucose solution: Dissolve 27.0 g of glucose in 25 mL of distilled water to prepare a 6 mol / L glucose solution. Prepare the standard curve of the glucose solution according to Table 1.

[0037] Table 1 Standard glucose solution

[0038] Test tube number Standard glucose solution / ml Sodium acetate buffer solution / ml Sodium hydroxide solution / ml DNS reagent / ml Glucose amount in test tube μmol 1 0 5 1 2 0 2 0.2 4.8 1 2 1.2 3 0.4 4.6 1 2 2.4 4 0.6 4.4 1 2 3.6 5 0.8 4.2 1 2 4.8 6 1.0 4.0 1 2 6.0 7 1.2 3.8 1 2 7.2 8 1.4 3.6 1 2 8.4

[0039] 1.3 Main culture media

[0040] The formula of the main culture media used in this experiment is shown in Table 2.

[0041] Table 2 Main culture media and their formulas

[0042] Culture medium name Culture medium formula Cellulose Congo red medium Microcrystalline cellulose 1.88 g, Congo red 1.0 g, gelatin 2.0 g, agar 20.0 g, distilled water 1000 mL. pH adjusted to 7.0 Sodium carboxymethyl cellulose medium (rescreening medium) <![CDATA[Sodium carboxymethyl cellulose 5.0 g, peptone 3.0 g, yeast extract 0.5 g, ammonium sulfate 2.0 g, K 2 HPO 4 4.0 g, CaCl 2 ·2H 2 O 0.3 g, MgSO 4 ·7H 2 O 0.3 g, distilled water 1000 mL, pH 7.2]]> LB liquid medium Tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, pH 7 Cellulose screening medium <![CDATA[Sodium carboxymethyl cellulose 15 g, yeast extract 1 g, NH 4 NO 3 1 g, MgSO 4 ·7H 2 O 0.5 g, KH 2 PO 4 1 g, agar 20 g, distilled water 1 L, pH 7]]> Lignin screening medium Lignin 1 g, beef extract 1 g, NaCl 1 g, peptone 2 g, distilled water 200 mL, natural pH

[0043] 1.4 Preservation methods of strains

[0044] (1) Slant preservation method: Inoculate the bacterial strain on the LB slant medium. After it covers the entire slant surface, place it in a refrigerator at 4 °C for low-temperature preservation. The preservation period is 1 - 3 months.

[0045] (2) Glycerol suspension preservation method: For bacterial strains that do not produce gas or mycelia, they need to be cultured in a mycelium liquid medium to obtain mycelia. After centrifuging to collect the mycelia, add 20% glycerol to make a suspension. When preserved in a refrigerator at -20 °C, the preservation period is 1 year; when preserved at -80 °C, the preservation period can reach more than 3 years.

[0046] 1.5 Experimental equipment

[0047] (1) Kits

[0048] The main kits used in this experiment are shown in Table 3.

[0049] Table 3 Main biochemical reagents and their sources

[0050] Reagent name Manufacturer Bacterial genomic DNA rapid extraction kit Beijing BioTeke Corporation 2×TaqPCR MasterMix Bomed Biotech Co., Ltd DNA marker Beijing ComWin Biotech Co., Ltd Agarose Promega Co. Ltd

[0051] (2) Instruments and equipment

[0052] The main instruments and equipment used in this experiment are shown in Table 4.

[0053] Table 4 Instruments and equipment

[0054] Name Model Manufacturer Biochemical incubator SHP - 450 Shanghai Jinghong Instrument Manufacturing Co., Ltd Laminar flow hood SW - CF - 1F Suzhou Sujie Purification Equipment Co., Ltd Electric thermostatic forced air drying oven DHG - 9140A Shanghai Yiheng Scientific Instruments Co., Ltd Electronic analytical balance AL204 Mettler - Toledo Instruments (Shanghai) Co., Ltd pH meter Delta 320 Mettler - Toledo Instruments Refrigerator - freezer BCD - 539WT Haier Co., Ltd Constant temperature shaker ZWYR - D2403 Shanghai Zhicheng Co., Ltd High - pressure steam sterilizer HVE - 2510 HIRAYAMA, Japan Constant temperature water bath 9112 PolyScience, USA Desktop refrigerated centrifuge Centrifuge 5417R Eppendorf, Germany PCR amplifier T1 Thermocycle Biometra, Germany Horizontal electrophoresis apparatus DYY - 8C Beijing Liuyi Instrument Factory Vortex oscillator HMQL - VORTEX - 5 Jiangsu Qilinbeier Instrument Manufacturing Co., Ltd, Haimen

[0055] 1.6 Data analysis software

[0056] The data of this experiment were analyzed for significant differences such as analysis of variance between samples and multiple comparisons (P < 0.05) using SPSS Version 22.

[0057] 2 Experimental methods and results

[0058] 2.1 Isolation and screening of cellulose-decomposing bacteria

[0059] Use a sterile mortar to grind 10 g of coral samples into a paste. Then weigh 1 g of the homogenate into a 50-ml conical flask containing 9 ml of sterile seawater and incubate it at 200 revolutions per minute and 37 °C for 30 minutes. Dilute it using the ten-fold serial dilution method to 10 -1 -10 -3Diluent. Take 100 μL of the diluent and evenly spread it on a Luria-Bertani (LB) agar plate, and culture it at 37 °C for 3 - 5 days. Isolate single colonies by repeated streaking, and then store them at -80 °C in 30% glycerol.

[0060] A total of 32 bacterial samples were isolated from stony corals. Then, one bacterial sample was spotted onto the center of a cellulose congo red medium by the spotting method. After culturing at a constant temperature of 30 °C for 7 days, observe whether a transparent circle appears around the colonies on the medium. Strains that produce a transparent circle indicate that they can produce cellulase. Measure the diameter (D) of the transparent circle and the diameter (d) of the colony on the plate, calculate the ratio of the two, and take the average value, denoted as EAI. The magnitude of the ratio can initially reflect the activity of the strain in producing cellulase. The results showed that the ratio of strain XZB-5 was the highest, reaching 4.91. Therefore, strain XZB-5 with the strongest cellulase activity was selected for identification and subsequent experiments.

[0061] 2.2 Strain classification and identification

[0062] (1) Culture characteristics and physicochemical properties

[0063] Refer to the "Manual for the Systematic Identification of Common Bacteria" to identify the physiological and biochemical characteristics of strain XZB-5, including: carbon source utilization experiment, nitrogen source utilization experiment, enzymological characteristic experiment, etc.

[0064] (1) Carbon and nitrogen source utilization experiments

[0065] Inoculate the strain to be tested into a medium containing different carbon sources and nitrogen sources, and observe its growth after culturing at 37 °C for 2 - 7 days, with a negative control set. The results are shown in Table 5. The 19 carbon sources and 9 nitrogen sources involved in the experiment can all be utilized by strain XZB-5.

[0066] (2) Hydrogen sulfide production experiment

[0067] Some strains can decompose sulfur-containing organic matter to produce hydrogen sulfide. Inoculate the strain on a hydrogen sulfide medium and culture it at 37 °C for 7 days. If the medium turns black, it indicates a positive result for hydrogen sulfide production; if the medium does not change color, it is negative. The results are shown in Table 5. Strain XZB-5 does not have the ability to produce hydrogen sulfide.

[0068] (3) Determination of enzymological characteristics

[0069] ① Esterase (Tween 20, Tween 40, Tween 80) experiment:

[0070] Inoculate the strain onto a urease medium and culture it at 37 °C for 2 days, then observe whether the medium changes color. Test the ability of the tested strain to produce urease. If the medium turns pink, it is positive; if it does not change color, it is negative.

[0071] ② Nitrate reduction

[0072] The test strain was inoculated into the nitrate reduction medium and cultured at 37 °C for 7 and 14 days, with the uninoculated medium as the control. Add a little of the culture solution cultured for 7 days and 14 days respectively into the test tubes, and drop one drop of Solution A and Solution B. The control was also added dropwise in the same way. When the solution turns pink, rose red, orange or brown, etc., it is positive for nitrate reduction; when there is no red color, add 1 or 2 drops of diphenylamine reagent. If it turns blue, the reduction is negative; if it does not turn blue, it is still regarded as positive.

[0073] The results showed that strain XZB-5 had multiple enzymatic activities, could reduce nitrate, and could produce esterase.

[0074] Table 5 Physiological and biochemical characteristics of strain XZB-5

[0075] Biochemical index Biochemical index Nitrate reduction + D - Mannitol ++ Hydrogen sulfide production - L - Arabinose +++ Tween 20 + Nitrogen source utilization Tween 40 + Phenylalanine - Tween 80 + Arginine +++ Carbon source utilization Cysteine - Sucrose ++ Methionine - D - Xylose +++ Histidine +++ D - Fructose ++ Ascorbic acid - Dextrin +++ Potassium nitrate +++ Soluble starch ++ Ammonium sulfate - Alkaline lignin + Anhydrous creatine - D - Galactose ++ L - Tyrosine - D - Sorbitol +++ L - Valine ++ Raffinose ++ Tryptophan - D - Mannitol +++ Acid hydrolyzed casein +++ D-Trehalose +++ Urea + Inositol +++ Glycine + Mannose +++ L-Methionine - Glucose +++ L-Glutamic acid - Salicin + C-Hydroxyproline + Melezitose +++ L-Asparagine +++ Rhamnose + Tyrosine -

[0076] (2)16S rDNA sequence analysis

[0077] The total DNA was extracted using the Bacterial Genomic DNA Rapid Extraction Kit (DP1301, Bioteke Corporation, Beijing, China). PCR amplification was performed using the universal primers for actinomycetes 16S rDNA (forward primer: 5’-AGAG TTTGATCCTGGCTCAG-3’, reverse primer: 5’-TACGGCTACCTTGTTACGACTT-3’) (Wang et al 2013). The specific reaction system is shown in Table 6, and the reaction program is shown in Table 7. The PCR product of strain XZB-5 was sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rDNA gene sequencing. The obtained gene sequence was compared using the BLAST software. At the same time, similar 16S rDNA gene sequences were searched in the GenBank and EzBioCloud databases. Strains with higher homology were selected for multiple comparison analysis. Finally, MEGA 7.0 was used to construct a phylogenetic tree using the Neighbor-Joining method (Tamura et al., 2011). The results are shown in Figure 1 , strain XZB-5 was closely related to the genus Bacillus ( Bacillus ), and phylogenetic analysis showed that it formed a unique branch (see Figure 3 ). It can be preliminarily judged that strain XZB-5 belongs to Bacillus genus.

[0078] Table 6 PCR amplification system

[0079] Reaction system Volume Template DNA 1 μL 2x Taq PCR MasterMix 12.5 μL Forward primer 0.5 μL Reverse primer 0.5 μL ddH2O 10.5 μL Total volume 25 μL

[0080] Table 7 PCR Amplification Program

[0081]

[0082] (3)Polyphasic Taxonomic Identification of Bacillus XZB-5

[0083] Pick a single colony of Bacillus XZB-5 and inoculate it into 100 mL of LB liquid medium. After culturing with shaking at 37 °C and 180 rpm for 2 d, collect the cells for total DNA extraction, and quantify the purified genomic DNA using a TBS-380 fluorometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

[0084] Genomic sequencing and assembly of Bacillus XZB-5 were performed using a next-generation sequencer (Shanghai Majorbio Bio-Pharm Technology Co., Ltd.). Genomic sequencing was carried out on the Illumina Hiseq × 10 platform. For DNA samples that passed quality inspection, fragments with an insert size of 400 bp were constructed for paired-end sequencing, with a single-end read length of 150 bp. Each sample provided at least 100× coverage depth of the original sequencing data volume of the genome, and finally, multiple genomic scaffolds were assembled.

[0085] Use the ANI calculation platform (https: / / www.ezbiocloud.net / tools / ANI) and the DNA-DNA hybridization calculation platform (https: / / ggdc.dsmz.de / ) to calculate the average nucleotide identity (ANI) and dDDH values (Yoon et al., 2017) between the scaffolds data of Bacillus XZB-5 and the scaffolds data of reference strains. The genomic data of reference strains were downloaded from the EzBioCloud public genomic database (https: / / www.ezbiocloud.net / search?tn=Nocardioides).

[0086] The results are as Figure 2 shown. From the BLAST results of the 16S rRNA sequence and the phylogenetic tree, strain XZB-5 was identified as the genus Bacillus ( Bacillus), by downloading the genomic data of standard strains from NCBI, since the genomic data of some strains were not uploaded, a total of 8 strains of bacteria were obtained through retrieval. The genomic data of these 8 groups were subjected to ANI and DDH tests with the genome of Bacillus XZB-5 one by one. ANI values ranged from 95% to 96%, and DNA-DNA hybridization values (DDH) from genomic nucleic acid sequences not higher than 70% are now considered the criteria for species delineation (Cui et al., 2021). By calculating the ANI and dDDH values between the genome of XZB-5 and those of the strains closely related to it, it was found that all these values were lower than the thresholds required to establish a new species (see Figure 2 B). Therefore, strain XZB-5 was identified as a new species of the genus Bacillus and named: Bacillus hainanensis XZB-5.

[0087] 2.4 Determination of cellulase activity and ligninase activity

[0088] Strain XZB-5 was cultured in a cellulase screening medium and a lignin screening medium for 7 days. The supernatant was collected daily and used as a crude enzyme solution to measure the activities of cellulases (exoglucanase, endoglucanase, β-glucosidase) and ligninases (manganese peroxidase, lignin peroxidase, laccase).

[0089] (1) Determination of cellulase activity

[0090] Endoglucanase activity: Using citrate buffer as a solvent, a 1% CMC substrate solution was prepared. In a 2 mL centrifuge tube, 100 μL of the substrate and 50 μL of the crude enzyme solution were added, mixed well, incubated in a water bath at 50 °C for 30 min, 200 μL of DNS solution was added to terminate the reaction, boiled for 5 min and cooled in ice water, and 1 mL of ddH2O was added and mixed well. 300 μL was taken and transferred to a 96-well microplate reader, and the absorbance was measured at 540 nm using a full-wavelength multifunctional microplate reader.

[0091] Exoglucanase activity: Using citrate buffer as a solvent, a 1% microcrystalline cellulose substrate solution was prepared. In a 2 mL centrifuge tube, 100 μL of the substrate and 50 μL of the crude enzyme solution were added, mixed well, incubated in a water bath at 50 °C for 30 min, 200 μL of DNS solution was added to terminate the reaction, boiled for 5 min and cooled in ice water, and 1 mL of ddH 2 O was added and mixed well. 300 μL was taken and transferred to a 96-well microplate reader, and the absorbance was measured at 540 nm using a full-wavelength multifunctional microplate reader.

[0092] β - Glucosidase activity: Using citrate buffer as a solvent, prepare a 1% salicin substrate solution. Add 100 μL of the substrate and 50 μL of the crude enzyme solution into a 2 mL centrifuge tube. After mixing, incubate in a water bath at 50 °C for 30 min. Add 200 μL of DNS solution to terminate the reaction, boil for 5 min, cool in ice water, and then add 1 mL of ddH2O and mix well. Take 300 μL and transfer it to a 96 - well microplate, and measure the absorbance at 540 nm using a full - wavelength multifunctional microplate reader.

[0093] The results are as Figure 3 shown in A. In the fermentation supernatant, the peak of cellulase activity was observed after 5 days. The endoglucanase activity was 11.63 U / mL, the exoglucanase activity was 9.74 U / mL, and the β - glucosidase activity was 6.82 U / mL. This indicates that strain XZB - 5 has good enzyme activities such as endoglucanase, exoglucanase, and β - glucosidase, suggesting that this strain can effectively degrade cellulose and hemicellulose.

[0094] (2) Determination of lignin - degrading enzyme activity

[0095] Determination of lignin peroxidase activity: Using the veratryl alcohol (VA) substrate method, 1 enzyme activity unit (U) is defined as the amount of enzyme required to oxidize 1 μmol of veratryl alcohol per minute. The reaction system is 8 mL, including 6 mL of 50 mmol / L sodium tartrate buffer (pH 3.0), 0.8 mL of 10 mmol / L VA, 1 mL of the crude enzyme solution. Add 0.2 mL of 10 mmol / L H2O2 to initiate the reaction under the condition of a 30 °C water bath, and measure the change in absorbance at 310 nm within the first 3 min of the reaction.

[0096] Determination of laccase activity: Using the ABTS substrate method, 1 enzyme activity unit (U) is defined as the amount of enzyme required to oxidize 1 μmol of ABTS per minute. The reaction system is 6 mL, including 3.6 mL of 50 mmol / L acetic acid - sodium acetate buffer solution (pH 4.0), 1.6 mL of 1 mmol / L ABTS, 0.8 mL of the crude enzyme solution. Measure the change in absorbance at 420 nm within the first 3 min of the reaction under the condition of a 30 °C water bath.

[0097] Determination of manganese peroxidase activity: Using the MnSO 4 substrate method, 1 enzyme activity unit (U) is defined as the amount of enzyme required to oxidize 1 μmol of MnSO 4 per minute. The reaction system is 8 mL, including 5 mL of 50 mmol / L acetic acid - sodium acetate buffer solution (pH 4.5), 0.8 mL of 1.6 mmol / L MnSO 4, 2 mL of crude enzyme solution, and 0.2 mL of 1.6 mmol / L H 2 O 2 were added to initiate the reaction, and the change in absorbance at 240 nm within the first 3 min before the reaction was measured.

[0098] Enzyme activity (U / mL) = (absorbance variable per minute of the sample - absorbance change per minute of the blank) * 1000000 * a * Ɛ * V

[0099] where: a - dilution factor of the sample; Ɛ is the extinction coefficient; V - volume of the reaction enzyme solution (mL).

[0100] Ɛ 310 = 9300 L / (mol·cm), Ɛ 420 = 36000 L / (mol·cm), Ɛ 240 = 11590 L / (mol·cm).

[0101] The results are as Figure 3 shown in B. The XZB-5 strain showed high lignin degradation activity. The activities of these three lignin-degrading enzymes initially increased and then decreased. The activities of lignin peroxidase and laccase reached peaks of 9.94 U / mL and 3.67 U / mL on the 3rd day, respectively, while the activity of manganese peroxidase reached its peak of 7.29 U / mL on the 4th day. This indicates that the strain can not only effectively degrade cellulose and hemicellulose but also lignin.

[0102] 2.4 Evaluation of the degradation ability of strain XZB-5 on various agricultural and forestry by-products

[0103] According to the method described by Srivastava et al. (2022), the degradation ability of strain XZB-5 on nine agricultural residues such as eucalyptus bark, peanut shells, rice straw, soybean meal, corn bran, rice husks, rape straw, wheat straw, and banana straw was analyzed. After drying all samples at 105 °C for 5 h, they were ground and passed through a 60-mesh sieve. 5 g of the selected residue was placed into a 250-mL conical flask with a water content of 70%. 0.5 mL of the XZB-5 strain spore suspension (108 CFU / mL) was inoculated into the conical flask and incubated at 37 °C and 180 rpm for 5 days. After centrifugation at 8000 rpm and 4 °C for 10 min, the supernatant was collected as the crude enzyme solution, and then the activities of the secreted cellulase and ligninase were evaluated.

[0104] The results are as Figure 3As shown in C and 3D, in the endoglucanase test, it was found that eucalyptus bark was the most effective carbon source, producing the highest activity of 5.88 ± 0.74 U / g. When banana straw was used as the fermentation substrate, the maximum activities of exoglucanase and β-glucosidase were measured to be 2.56 ± 0.04 U / g and 2.83 ± 0.08 U / g, respectively. When rice husk was used as the fermentation substrate, the lowest cellulase activities were obtained, with β-glucosidase activity of 0.02 ± 0.01 U / g, endoglucanase activity of 0.04 ± 0.01 U / g, and exoglucanase activity of 0.05 ± 0.02 U / g. For lignin enzymes, in the presence of eucalyptus bark and banana straw, the highest activities of manganese peroxidase, lignin peroxidase, and laccase were 3.64 ± 0.10 U / g, 5.21 ± 0.53 U / g, and 2.01 ± 0.14 U / g, respectively.

[0105] 2.5 Composting experiment of strain XZB-5

[0106] The analysis results of the ability of strain XZB-5 to degrade various agricultural wastes showed that eucalyptus bark exhibited the highest degradation effect. Therefore, eucalyptus bark was selected as the material for the composting experiment. It should be understood that peanut shells, rice straw, soybean meal, corn bran, rice husks, rapeseed straw, wheat straw, and banana straw can also be used for the composting experiment. The composting experiment was carried out from July 14 to September 1, 2023, at the experimental site of the Chinese Academy of Tropical Agricultural Sciences in Danzhou, Hainan Province, China. The eucalyptus bark was cut into sections about 3 cm long and then divided into two piles of 1 cubic meter, including non-inoculated (control) and inoculated with strain XZB-5 (treatment). The initial C / N ratio was adjusted to 25 with molasses. The initial water content was adjusted to 60% with sterile water to enhance the decomposition of organic matter. The compost was manually turned and mixed every 5 days to ensure sufficient oxygen supply for the microorganisms. The composting was maintained for 50 days, and samples were collected every 10 days. The initial mixture without inoculating any microorganisms was labeled as CK0. The collected samples were divided into two parts: one part was stored at -80 °C for analyzing the bacterial community, and the other part was kept at 4 °C for physicochemical evaluation.

[0107] (1) Physicochemical property analysis

[0108] The temperature of the compost was measured every day at 9 am and 5 pm using a thermometer, and the actual temperature was determined by the average of these two readings. At the same time, the ambient temperature was also recorded. An electrical conductivity meter (INESA, Shanghai, China) was used to measure the electrical conductivity. The germination index (GI) was evaluated by placing corn seeds in the water extract of fresh compost samples (1:10, m:v). Each treatment included 20 seeds, with 3 biological replicates.

[0109] The results are as Figure 4As shown. Temperature is the most critical factor in controlling the composting process. It regulates the transformation of organic matter and serves as an indicator of changes in microbial activity during the composting cycle. This study revealed that inoculation with strain XZB-5 could induce heat activation during the thermophilic stage, reaching a peak temperature of 71 °C on the 7th day, exceeding that of the control group (60 °C on the 10th day) (see Figure 4 B). During the entire composting process, electrical conductivity (EC), as a major indicator, showed a consistent fluctuation pattern in both the control group and the treatment group: it initially increased, then decreased, and then increased again during the composting process (see Figure 4 C). A seed germination rate value higher than 80% usually indicates that the compost has reached a mature level and is harmless to plant growth (Jia et al., 2023). In this study, the seed germination rates of the two groups were low at the initial stage of composting (the 10th day of composting), with values of 43.33% and 26.67% respectively (see Figure 4 D). On the 30th day of composting, the seed germination rate of the control group was still lower than 80%, while that of the treatment group reached 86.67%. In the final compost, the seed germination rate values of the two groups were 83.33% and 98.33% respectively, indicating that the compost had reached a mature level. Therefore, inoculation with strain XZB-5 can accelerate the transformation of compost products and shorten the composting period.

[0110] (2) Analysis of cellulase and ligninase activities

[0111] To measure the enzyme activities, 1 g of fresh compost sample was mixed with 10 mL of 50 mM citrate buffer (pH 6.5), and then incubated at 4 °C and 180 rpm for 1 hour. After centrifugation at 8000 rpm and 4 °C for 10 minutes, the supernatant was collected as the crude enzyme solution to determine the activities of cellulase (exoglucanase, endoglucanase, β-glucosidase) and ligninase (manganese peroxidase, lignin peroxidase, laccase). The measurement method was referred to Section 2.3 above.

[0112] The results are as Figure 5As shown in the figure. The enzyme activities of exoglucanase, endoglucanase, β-glucosidase, manganese peroxidase, lignin peroxidase and laccase during the composting process all followed a similar trend, that is, initially increasing and then decreasing during the composting process. The enzyme activities reached their maximum values on the 30th day of composting. As cellulases involved in cellulose degradation, the highest activities of endoglucanase, exoglucanase and β-glucosidase in the treatment group were 9.99 U / g, 8.14 U / g and 4.88 U / g respectively, which were significantly higher than those in the control group. For lignin-degrading enzymes, the maximum activities of manganese peroxidase, lignin peroxidase and laccase in the treatment group were 11.43 U / g, 15.77 U / g and 5.55 U / g respectively. Through the changes in the activities of manganese peroxidase, lignin peroxidase and laccase during the cooling and maturation stages of composting, it can be clearly seen that the levels in the treatment group were significantly higher than those in the control group. Therefore, inoculating with strain XZB-5 can accelerate the degradation of lignocellulose in compost products and speed up the conversion of resources.

[0113] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A Hainan Bacillus, which is a new species of Bacillus, characterized in that: Named Bacillus hainanensis XZB-5 is registered and deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC NO: 64531.

2. The fermentation liquid of the Hainan Bacillus according to claim 1.

3. A bacterial agent containing the Hainan Bacillus according to claim 1.

4. A lignin degrading enzyme preparation, characterized in that A fermentation liquid containing the Hainan Bacillus according to claim 1.

5. Use of the Hainan Bacillus according to claim 1, or the fermentation liquid according to claim 2, or the bacterial agent according to claim 3, or the lignin degrading enzyme preparation according to claim 4 in degrading cellulose and / or lignin.

6. Use of the Hainan Bacillus as claimed in claim 1, or the fermentation broth as claimed in claim 2, or the bacterial agent as claimed in claim 3, or the lignin degrading enzyme preparation as claimed in claim 4 in the preparation of preparations producing exoglucanase, and / or endoglucanase, and / or β-glucosidase, and / or manganese peroxidase, and / or lignin peroxidase, and / or laccase.

7. Use of the Hainan Bacillus according to claim 1, or the fermentation liquid according to claim 2, or the bacterial agent according to claim 3, or the lignin degrading enzyme preparation according to claim 4 in degrading agricultural residues; or The use of the Hainan Bacillus as claimed in claim 1, or the fermentation liquid as claimed in claim 2, or the bacterial agent as claimed in claim 3, or the lignin degrading enzyme preparation as claimed in claim 4 in degrading eucalyptus bark, and / or peanut shells, and / or rice straw, and / or soybean meal, and / or corn bran, and / or rice husks, and / or rape straw, and / or wheat straw, and / or banana straw.

8. Use of the Hainan Bacillus according to claim 1, or the fermentation liquid according to claim 2, or the bacterial agent according to claim 3, or the lignin degrading enzyme preparation according to claim 4 in improving seed germination rate.

9. Use of the Hainan Bacillus according to claim 1, or the fermentation liquid according to claim 2, or the bacterial agent according to claim 3, or the lignin degrading enzyme preparation according to claim 4 in the preparation of fertilizers.

10. A fertilizer containing the Hainan Bacillus according to claim 1, or the fermentation liquid according to claim 2, or the bacterial agent according to claim 3, or the lignin-degrading enzyme preparation according to claim 4.

Images