Bacillus amyloliquefaciens and its applications

Abstract

The present invention discloses Bacillus saccharolyticus for dissolving phosphorus and its applications. The Bacillus saccharolyticus for dissolving phosphorus provided by the present invention ( Saccharibacillus phosphatilytica ) S22909 has a registration number of CGMCC No. 25247 in the China General Microbiological Culture Collection Center. The Bacillus saccharolyticus S22909 provided by the present invention represents a new species of the genus Bacillus saccharolyticus. Through the detection of alkaline phosphatase activity, it is proved that this strain has alkaline phosphatase activity and can convert insoluble phosphorus in the soil into soluble phosphorus for plant absorption and utilization, and has broad application prospects in degrading organic phosphorus and promoting plant growth. At the same time, this strain has the ability to degrade cellulose and has broad application prospects in cellulose degradation. Therefore, the strain of the present invention can be used for the development of phosphorus-dissolving bacterial fertilizers and soil improvement in different aspects.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and particularly to Saccharibacillus phosphorolyticus and its applications. Background Art

[0002] The genus Saccharibacillus (Rivas R, García-Fraile P, Zurdo- JL, Mateos PF, Martínez-Molina E, Bedmar EJ, Sánchez-Raya J, Velázquez E (2008). Saccharibacillus sacchari gen. nov., sp. nov., isolated from sugar cane. Int JSyst Evol Microbiol 58, 1850-1854.) Established in 2008, it belongs to the family Paenibacillaceae (De Vos P, Ludwig W, Schleifer K-H, Whitman WB (2009). Family IV. Paenibacillaceae fam. nov. In: De Vos P, Garrity GM, Jones D, Krieg NR, Ludwig W, Rainey FA, K.-H. S, Whitman WB (eds), Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 3 (The Firmicutes), Springer, New York, p. 269.).So far, five validly described species have been included in the genus Saccharibacillus: S. endophyticus, an endophyte isolated from cotton plants (Kampfer P, Busse HJ, Kleinhagauer T, McInroy JA, Glaeser SP (2016). Saccharibacillus endophyticus sp. nov., an endophyte of cotton. Int J Syst Evol Microbiol 66, 5134-5139.); S. qingshengii, isolated from a lead-cadmium tailing soil sample (Han H, Gao S, Wang Q, He LY, Sheng XF (2016). Saccharibacillus qingshengii sp. nov., isolated from a lead-cadmium tailing. Int J Syst Evol Microbiol 66, 4645-4649.); S. sacchari, isolated from the extracellular fluid of sugarcane (Rivas R, Garcia-Fraile P, Zurdo-Pineiro JL, Mateos PF, Martinez-Molina E, Bedmar EJ, Sanchez-Raya J, Velazquez E (2008). Saccharibacillus sacchari gen. nov., sp. nov., isolated from sugar cane. Int J Syst Evol Microbiol 58, 1850-1854.), and S. deserti (Sun JQ, Wang XY, Wang LJ, Xu L, Liu M, Wu XL (2016). Saccharibacillus deserti sp. nov., isolated from desert soil. Int J Syst Evol Microbiol 66, 623-627.) and S. kuerlensis (Yang SY, Liu H, Liu R, Zhang KY, Lai R (2009). Saccharibacillus kuerlensis sp. nov., isolated from a desert soil. Int J Syst Evol Microbiol 59, 953-957.), both isolated from desert soil samples.In recent years, with the development of high-throughput sequencing technology, the high-throughput sequencing technology of 16S rRNA gene developed by Illumina can help to quickly and accurately analyze the microbial community structure and diversity information in samples, and more and more researchers are applying this technology to study related problems. Bziuk et al. studied the microbial community in barley seeds by 16S rRNA gene amplicon sequencing method and found that Bacillus saccharolyticus strains were one of the dominant bacterial groups in the microbial community of this habitat (Bziuk N, Maccario L, Straube B, Wehner G, SJ, Schikora A, Smalla K(2021). The treasure inside barley seeds: microbial diversity and plant beneficial bacteria. Environ Microbiome 16, 20.).

[0003] Bacillus saccharolyticus has its characteristic morphology and chemotaxonomic characteristics: the cells of the genus members are all rod-shaped and can form endospores. The catalase and oxidase reactions are both negative. The main menaquinone component is MK-7, and the main polar lipid components are diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and an unknown aminophospholipid (APG). In addition, it may also contain unknown glycolipid (GL) and unknown phosphoglycolipid (GPL). The main fatty acids are anteiso-C 15:0 and C 16:0 .

[0004] Phosphorus is an essential nutrient element for plant growth and development and is indispensable for important life activities such as photosynthesis and respiration in plants. Most of the phosphorus in the soil exists in the form of insoluble Fe-P, Al-P and occluded phosphorus that plants cannot directly absorb and utilize, resulting in rich total phosphorus content and scarce available phosphorus in the soil. Phosphorus-solubilizing bacteria can convert insoluble phosphorus in the soil into available phosphorus, thereby improving the phosphorus nutrition of plants and promoting plant growth. Although researchers have isolated many strains of phosphorus-solubilizing bacteria from various plant rhizosphere soil habitats, there are few reports on Bacillus saccharolyticus strains.

[0005] Cellulose is one of the most abundant renewable resources in the world, providing a rich raw material for the development of renewable biofuels. However, the saccharification of cellulose has always been a bottleneck in its utilization process. Endoglucanase produced by microorganisms is a key enzyme system for cellulose degradation and conversion, which can degrade cellulose into cellooligosaccharides. Therefore, endoglucanase-producing bacteria can play an important role in the saccharification process of cellulose. At present, there are few reports on the endoglucanase activity of strains belonging to the genus Saccharibacillus. Summary of the Invention

[0006] The object of the present invention is to provide a new species of the genus Saccharibacillus and its application.

[0007] In a first aspect, the present invention claims protection for a new species of the genus Saccharibacillus.

[0008] The new species of the genus Saccharibacillus claimed by the present invention is specifically Saccharibacillus phosphatilytica S22909, and its registration number in the China General Microbiological Culture Collection Center is CGMCC No. 25247.

[0009] The Saccharibacillus phosphatilytica S22909 is a Gram-positive bacterium with peritrichous flagella and is motile. Its rod shape has dimensions of (0.4-1) μm × (2-5) μm, and it can produce ellipsoidal endospores. When cultured on tryptic soy agar medium at 28°C for 48 h, it can form white to milky white colonies. The pH tolerance range of strain S22909 is 6.0-8.0, and the optimal growth pH value is 7.0. The oxidase and catalase reactions are negative. It can hydrolyze starch and urea and has the ability to reduce nitrate. It can utilize dextrin, D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, D-turanose, stachyose, D-raffinose, α-D-lactose, D-melibiose, β-methyl-D-glucoside, D-salicin, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetylneuraminic acid, α-D-glucose, D-mannose, D-fructose, D-galactose, L-fucose, L-rhamnose, inosine, D-sorbitol, D-mannitol, pectin, inositol, glycerol, D-arabitol, L-galactonolactone, D-gluconic acid, D-glucuronic acid, methyl pyruvate, L-lactic acid, α-ketoglutaric acid, bromosuccinic acid, α-ketobutyric acid, acetoacetic acid, propionic acid, D-galacturonic acid, and Tween 40 as the sole carbon source. It has alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, and β-glucosidase activities. The 16S rRNA sequence of this strain is shown in SEQ ID No.1.

[0010] In a second aspect, the present invention claims protection for a culture.

[0011] The culture claimed by the present invention is the culture of the Saccharibacillus phosphatilytica S22909 described in the first aspect above, specifically the substance obtained by culturing the Saccharibacillus phosphatilytica S22909 in a bacterial medium.

[0012] In the above-mentioned culture, the substance includes the Saccharibacillus phosphatilytica S22909 (the bacterium itself) and the metabolites of the Saccharibacillus phosphatilytica S22909.

[0013] In the above-mentioned culture, the bacterial medium can be a solid medium or a liquid medium.

[0014] The term "culture" generally refers to a liquid or solid medium with a microbial population after artificial inoculation and cultivation. That is, a product obtained by growing and / or amplifying microorganisms, which can be a pure biological culture of microorganisms or can contain a certain amount of medium, metabolites or other components produced during the cultivation process. The term "culture" also includes subcultures obtained by subculturing microorganisms, which can be a culture of a certain generation or a mixture of several generations.

[0015] In a specific embodiment of the present invention, the bacterial medium is specifically tryptone soy agar medium.

[0016] In a third aspect, the present invention claims protection for a metabolite.

[0017] The metabolite claimed by the present invention is the metabolite of Saccharibacillus phosphatilytica S22909 described in the first aspect above.

[0018] The term "metabolite" refers to primary metabolites and / or secondary metabolites produced during the metabolism of microorganisms. Primary metabolism refers to the process by which microorganisms absorb various nutrients from the outside world and generate substances and energy for maintaining life activities through catabolism and anabolism. The products of primary metabolism are primary metabolites, such as monomers of monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, etc., and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, lipids, etc. Secondary metabolism refers to the process by which microorganisms synthesize some substances with no clear function for the life activities of microorganisms during a certain growth period using primary metabolites as precursors. The products of secondary metabolism are secondary metabolites, most of which are compounds with relatively complex molecular structures. According to their functions, they can be classified into types such as antibiotics, hormones, alkaloids, toxins, etc.

[0019] In a fourth aspect, the present invention claims protection for a bacterial agent.

[0020] The bacterial agent claimed by the present invention contains Saccharibacillus phosphatilytica S22909 described in the first aspect above, the culture described in the second aspect above, and / or the metabolite described in the third aspect above.

[0021] The bacterial agent is a bacterial agent for hydrolyzing cellulose and / or degrading organic phosphorus.

[0022] In the above-mentioned bacterial agent, in addition to the active ingredient, the bacterial agent further contains a carrier. The carrier can be a carrier commonly used in the pesticide field and biologically inert. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material or a polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material can be at least one of corn flour, bean flour and starch; the polymer compound can be polyvinyl alcohol and / or polyglycol; the liquid carrier can be an organic solvent, a vegetable oil, a mineral oil or water; the organic solvent can be decane and / or dodecane.

[0023] In the above-mentioned bacterial agent, the dosage form of the bacterial agent can be various dosage forms, such as a liquid agent, an emulsion, a suspension, a powder, a granule, a wettable powder or a water-dispersible granule.

[0024] According to needs, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added to the bacterial agent.

[0025] In the fifth aspect, the present invention claims the use of the phosphorus-solubilizing Saccharibacillus phosphatilytica S22909 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above in any of the following:

[0026] (A1) Degrading cellulose;

[0027] (A2) Preparing a product for degrading cellulose;

[0028] (A3) Preparing cellulase;

[0029] (A4) Preparing a product with cellulase activity;

[0030] (A5) Preparing a product with endoglucanase activity;

[0031] (A6) Degrading organic phosphorus;

[0032] (A7) Preparing a product for degrading organic phosphorus;

[0033] (A8) Preparing a product with alkaline phosphatase activity.

[0034] In the sixth aspect, the present invention claims the use of the phosphorus-solubilizing Saccharibacillus phosphatilytica S22909 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above in any of the following:

[0035] (B1) Promote plant growth;

[0036] (B2) Develop phosphate-solubilizing bacterial fertilizers;

[0037] (B3) Environmental governance;

[0038] (B4) Soil improvement;

[0039] (B5) Biofuel development.

[0040] In the seventh aspect, the present invention claims to protect a product for degrading cellulose.

[0041] The product for degrading cellulose claimed by the present invention has an active ingredient which is the Saccharibacillus phosphatilytica S22909 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0042] In the eighth aspect, the present invention claims to protect a product having cellulase activity.

[0043] The product having cellulase activity claimed by the present invention has an active ingredient which is the Saccharibacillus phosphatilytica S22909 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0044] In the ninth aspect, the present invention claims to protect a product for degrading organic phosphorus.

[0045] The product for degrading organic phosphorus claimed by the present invention has an active ingredient which is the Saccharibacillus phosphatilytica S22909 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0046] In the tenth aspect, the present invention claims to protect a product having alkaline phosphatase activity.

[0047] The product having alkaline phosphatase activity claimed by the present invention has an active ingredient which is the Saccharibacillus phosphatilytica S22909 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0048] In the eleventh aspect, the present invention claims a method for degrading cellulose.

[0049] The method for degrading cellulose claimed by the present invention may include the following steps: treating a sample to be treated with the Saccharibacillus phosphatilytica S22909 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0050] In the twelfth aspect, the present invention claims a method for degrading organic phosphorus.

[0051] The method for degrading organic phosphorus claimed by the present invention may include the following steps: treating a sample to be treated with the Saccharibacillus phosphatilytica S22909 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0052] In the thirteenth aspect, the present invention claims the use of the Saccharibacillus phosphatilytica S22909 described in the first aspect above in the preparation of the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0053] Experimental results show that there are many significant differences between the Saccharibacillus phosphatilytica S22909 provided by the present invention and existing strains of the genus Saccharibacillus in terms of phenotypic, physiological and biochemical, and cytochemical characteristics. Meanwhile, phylogenetic analysis at the gene level further shows that the strain S22909 can be distinguished from each valid species of the existing genus Saccharibacillus, fully proving that the strain S22909 of the present invention represents a new species of the genus Saccharibacillus, named Saccharibacillus phosphatilytica. At the same time, through the detection of alkaline phosphatase activity, it is also proved that the strain of the present invention has alkaline phosphatase activity, can convert insoluble phosphorus in the soil into soluble phosphorus for plant absorption and utilization, and has broad application prospects in degrading organic phosphorus and promoting plant growth; meanwhile, this strain has the ability to degrade cellulose and has broad application prospects in cellulose degradation. Therefore, the strain of the present invention can be used in the future for the development of various aspects such as phosphorus-solubilizing bacterial fertilizers, environmental governance, soil improvement production, and biofuel development.

[0054] Depository Instructions

[0055] Classification and Naming: Saccharibacillus phosphatilytica;

[0056] Biological Material for Reference: S22909;

[0057] Depository Institution: General Microbiological Center of China Committee for Culture Collection of Microorganisms;

[0058] Abbreviation of Depository Institution: CGMCC;

[0059] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0060] Date of Deposit: July 7, 2022;

[0061] Registration Number in the Depository Center: CGMCC No. 25247. Description of the Drawings

[0062] Figure 1 It is a photo of the cell morphology of strain S22909 cultured on tryptic soy agar medium at 28°C for 48 h.

[0063] Figure 2 It is a picture for screening the phosphorus-solubilizing ability of strain S22909.

[0064] Figure 3 It is a phylogenetic tree constructed based on the 16S rRNA gene sequences of strain S22909 and related species in the family Paenibacillaceae (the phylogenetic tree uses Lactobacillus delbrueckii subsp. lactis DSM 20072 T (GenBank accession no. M58823) as the outgroup). Detailed Embodiments

[0065] The present invention will be further described in detail below in conjunction with the detailed embodiments. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0066] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0067] Isolation and Identification of Saccharibacillus phosphatilytica S22909 in Example 1

[0068] I. Isolation of Strain S22909

[0069] The strain S22909 of the present invention was isolated from a rhizosphere soil sample in Xinping Yi and Dai Autonomous County, Yuxi City, Yunnan Province. The specific isolation operation is as follows:

[0070] Preparation of soil suspension: Add 2 g of soil sample into 18 mL of a sterile mixed solution containing 0.1% sodium pyrophosphate and 0.85% sodium chloride, place it in a shaker at 28 °C with 180 rpm, and shake for 40 min to fully suspend the soil particles. Gradient dilute the formed soil suspension to 10 -4 for standby.

[0071] Isolation medium: Soluble starch 10 g·L -1 , dipotassium hydrogen phosphate 1 g·L -1 , magnesium sulfate heptahydrate 1 g·L -1 , ammonium sulfate 2 g·L -1 , calcium carbonate 2 g·L -1 , ferrous sulfate heptahydrate 0.001 g·L -1 , manganese chloride 0.001 g·L -1 , zinc sulfate 0.001 g·L -1 , sodium chloride 1 g·L -1 , glycerol 12.5 mL·L -1 , arginine 2 g·L -1 , marine micro salt 0.38 g·L -1 , agar 15 g·L -1 ; pH 7.

[0072] Isolation method: Spread the above-diluted soil suspension on the isolation medium plate and culture it at 28 °C for 3 weeks. Pick out well-grown single colonies and culture them on a PYG slant medium (formula: peptone 3 g·L -1 , yeast extract 5 g·L -1 , glycerol 10 g·L -1 , betaine 1.25 g·L -1 , sodium pyruvate 1.25 g·L -1 , agar 15 g·L -1 ; pH 7) to obtain pure cultures for subsequent research. At the same time, the obtained pure strains were cryopreserved in liquid nitrogen and at -80 °C with 20% (v / v) glycerol as a cryoprotectant. Obtain a strain that is related to the closest reference strains S.kuerlensis HR1 T and S.sacchari DSM 19268T The strains with a similarity of 96.4% are numbered S22909.

[0073] II. Identification of Strain S22909

[0074] Strain S22909 was grown on tryptic soy broth medium (Solarbio) at 28 °C for morphological, physiological and biochemical, cytochemical and genetic level studies. Other special cases will be described.

[0075] 1. Observation of cell morphology and detection of physiological and biochemical characteristics of strain S22909

[0076] After strain S22909 was cultured on tryptic soy agar medium at 28 °C for 48 h, transmission electron microscopy (JEOL, JEM-1400) was used for cell morphology observation. The growth temperature detection range of strain S22909 was 4, 10, 28, 30, 32, 37, 42 and 45 °C; the growth salt concentration (NaCl) detection range was 0, 1, 3, 5, 7, 10% (g / 100 mL); the growth pH detection range was 8 gradients between pH 4 - 11 (4, 5, 6, 7, 8, 9, 10, 11). The physiological and biochemical characteristics of the strain were detected using API50CH, API ZYM, and BiOLOG GEN III carbon source detection kits. Other strain physiological characteristics, including Gram staining property, oxygen requirement, catalase activity, oxidase activity, gelatin hydrolysis activity, starch hydrolysis activity and cellulose hydrolysis activity, mainly referred to "Actinomycete System Identification Manual" (Xu L H (2007). Actinomycete systematics: principles, methods and practices. Beijing: Science Press, 93 - 108.).

[0077] The identification results showed that strain S22909 was a Gram-positive bacterium with peritrichous flagella and motility ( Figure 1) with a rod shape size of (0.4 - 1) μm × (2 - 5) μm, capable of producing ellipsoidal endospores. This strain is cultured on tryptone soy agar medium at 28°C for 48 h, and can form white to milky white colonies. The pH tolerance range of strain S22909 is 6.0 - 8.0, and the optimal growth pH value is 7.0. The oxidase and catalase reactions are negative. It can hydrolyze starch and urea, and has the ability to reduce nitrate. It can utilize dextrin, D - maltose, D - trehalose, D - cellobiose, gentiobiose, sucrose, D - turanose, stachyose, D - raffinose, α - D - lactose, D - melibiose, β - methyl - D - glucoside, D - salicin, N - acetyl - D - glucosamine, N - acetyl - D - galactosamine, N - acetylneuraminic acid, α - D - glucose, D - mannose, D - fructose, D - galactose, L - fucose, L - rhamnose, inosine, D - sorbitol, D - mannitol, pectin, inositol, glycerol, D - arabitol, L - galactono - 1,4 - lactone, D - gluconic acid, D - glucuronic acid, methyl pyruvate, L - lactic acid, α - ketoglutaric acid, bromosuccinic acid, α - ketobutyric acid, acetoacetic acid, propionic acid, D - galacturonic acid and Tween 40 as the sole carbon source. It has alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, α - chymotrypsin, acid phosphatase, naphthol - AS - BI - phosphohydrolase, β - galactosidase, β - glucosidase activities. The differential characteristics between strain S22909 and the representative strains of related species in the genus Saccharibacillus are shown in Table 1.

[0078] Table 1. Differential characteristics between strain S22909 and related strains in the genus Saccharibacillus

[0079]

[0080]

[0081]

[0082] Note: In the table, + indicates positive, - indicates negative, and ND indicates that no relevant data was searched.

[0083] As can be seen from the results shown in Table 1, there are significant differences in some physiological and biochemical characteristics between the strain S22909 of the present invention and the published related strains in the genus Saccharibacillus.

[0084] 2. Detection of the cytochemical characteristics of strain S22909

[0085] The fatty acids, quinone types, polar lipids and other cytochemical components of strain S22909 were detected by GC (gas chromatography), HPLC (high performance liquid chromatography) and TLC (thin layer chromatography) (Sasser M. Identification of bacteria by gasghromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDIinc; 1990. Minnikin DE, O’Donnell AG, Goodfellow M, Alderson G, Athalye M et al. Anintegrated procedure for the extraction of bacterial isoprenoid quinones andpolar lipids. J Microbiol Methods 1984;2:233-241.).

[0086] The experimental results showed that the main fatty acid components of strain S22909 of the present invention were anteiso-C 15:0 (30.4%), iso-C 15:0 (39.7%), C 16:0 (9.5%). Among them, the main components anteiso-C 15:0 and C 16:0 were consistent with other species of the genus Sporolactobacillus. However, strain S22909 of the present invention had unique features different from other related species, and iso-C 15:0 was its unique component. In addition, the main polar lipid components of strain S22909 of the present invention included diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG). See Table 1. The main menaquinone component of strain S22909 of the present invention was MK-7, which was consistent with the main menaquinone component of the genus Sporolactobacillus. The above results all indicated that strain S22909 was a new species of the genus Sporolactobacillus.

[0087] 3. Determination of alkaline phosphatase activity of strain S22909

[0088] Using lecithin as the only phosphorus source in the plate, the alkaline phosphatase activity of strain S22909 was screened on the plate. The specific steps were as follows: Prepare an organic phosphorus solid screening medium (formula: glucose 3 g·L -1 , ammonium sulfate 0.5 g·L -1 , sodium chloride 0.3 g·L -1 , potassium chloride 0.3 g·L -1 , ferrous sulfate heptahydrate 0.03 g·L -1, Manganese sulfate heptahydrate 0.03 g·L -1 , Calcium carbonate 5 g·L -1 , Lecithin 1 g·L -1 , Agar 15 g·L -1 ; (pH 7), autoclaved at 115 °C for 30 min. Subsequently, pick a single colony of the strain S22909 in the logarithmic growth phase and inoculate it on the organic phosphorus solid screening medium. After culturing at 28 °C for 4 days, measure the diameter (d) of the bacterial colony and the diameter (D) of the phosphorus solubilizing circle respectively, and calculate the solubility index (D / d).

[0089] The experimental results show that ( Figure 2 ), the strain S22909 of the present invention can produce a phosphorus solubilizing circle on the organic phosphorus solid screening medium, its colony diameter is 5.0 mm, the transparent circle diameter is 17.0 mm, and the solubility index is 3.4.

[0090] At the same time, perform liquid screening on the alkaline phosphatase activity of the strain S22909. Inoculate the strain S22909 in the logarithmic growth phase into 20 mL of the organic phosphorus liquid medium, and use the non-inoculated blank medium as the control group. After culturing at 28 °C for 14 days, centrifuge to take the supernatant and measure the organic phosphorus concentration in the culture solution, and use the blank group as the control to calculate the PO4 3+ (mg / L) concentration change (represented by △PO4 3+ ). Prepare a standard curve according to the phosphate standard solution. M0 is the PO4 3+ concentration detected in the blank control group, and M1 is the PO4 3+ concentration detected in the experimental group.

[0091] △PO4 3+ concentration = M1 - M0

[0092] After calculation, the strain S22909 of the present invention has alkaline phosphatase activity, and the △PO4 3+ concentration value for degrading organic phosphorus is 0.40 mg / L.

[0093] In addition, use the diamond software to compare the genomic data of the strain S22909 with the eggNOG database (http: / / eggnog.embl.de / ) and the KEGG database (http: / / kobas.cbi.pku.edu.cn / home.do) respectively, and set the E value to be lower than e -5 . We searched for the alkaline phosphatase A gene (pho A) and the alkaline phosphatase E (pho E) gene from the genome of this strain. The specific information is shown in Table 2.

[0094] Table 2. Alkaline phosphatase genes in the genome of strain S22909

[0095]

[0096] 4. Detection of Endoglucanase Activity of Strains

[0097] The endoglucanase activity of strain S22909 was screened on a plate using sodium carboxymethyl cellulose (CMC-Na) as the sole C source in the plate (Reinhold-Hurek, B., Hurek. T., Claeyssens, M., van Montagu, M. (1993). Cloning, expression in Escherichia coli, and characterization of cellulolytic enzymes of Azoarcus sp., a root-invading diazotroph. J Bacteriol 175, 7056-7065.). The specific steps are as follows: Prepare the CMC-Na screening medium (formula: (NH4)2SO4 4 g·L -1 , NaCl 0.1 g·L -1 , MgSO4·7H20 0.1 g·L -1 , CaCl2 0.1 g·L -1 , yeast extract 0.5 g·L -1 , Fe(Ⅲ)EDTA 0.033 g·L -1 , CMC-Na 2 g·L -1 , agar 15 g·L -1 ; pH 7.0). Pick a single colony of strain S22909 in the logarithmic growth phase and inoculate it on the CMC-Na screening medium. After culturing for 48 h, use Congo red staining solution to detect the endoglucanase activity of the strain. The results showed that an obvious transparent circle was produced around strain S22909, indicating that strain S22909 has good cellulose hydrolysis activity. The same experimental method was used for re-screening verification and the same experimental results as the primary screening were found (Teather, R.M., Wood, P.J. (1982). Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 43, 777-780.).

[0098] In addition, the genomic data of strain S22909 was compared with the eggNOG database (http: / / eggnog.embl.de / ) and the KEGG database (http: / / kobas.cbi.pku.edu.cn / home.do) using diamond software, with the E value set below e -5 . Two copies of endoglucanase genes were searched from the genome of this strain, and both belonged to the GH5 endoglucanase genes. The specific information is shown in Table 3.

[0099] Table 3. Endoglucanase genes in the genome of strain S22909

[0100]

[0101] 5. Determination of the phylogenetic status of the strain

[0102] The genomic DNA of the strain S22909 of the present invention was extracted for sequencing, and the 16S rRNA gene sequence (SEQ ID No.1) was aligned online in the internationally authoritative bacterial taxonomy analysis database (http: / / www.ezbiocloud.net / ) (Kim, O.S., Cho, Y.J., Lee, K.,, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J (2012). Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716 - 721.). The results showed that the strain S22909 of the present invention had the highest similarity with the species of the genus Saccharibacillus, and the representative strains of the species with the highest pairwise sequence similarity were S. kuerlensis HR1 T (similarity 96.4%) and S. sacchari DSM 19268 T(The similarity is 96.4%), which is significantly lower than the defined threshold of 98.7% for different bacterial species (Stackebrandt E, Ebers J (2006). Taxonomic parameters revisited: tarnished gold standards. Microbiol Today 33, 152 - 155.). The average nucleotide identity (ANI value) of the whole - genome sequence of strain S22909 and that of closely related control bacteria was compared and calculated on EZbiocloud, and the results showed that strain S22909 and the closely related control bacterium S. kuerlensis HR1 T had an ANI ratio of 77.1%, and strain S22909 and the closely related control bacterium S. sacchari DSM 19268 T had an ANI ratio of 77.9%, both of which were lower than the defined threshold of 95.0% for different bacterial species (Yoon SH, Ha SM, Lim J, Kwon S, Chun J (2017). A large - scale evaluation of algorithms to calculate average nucleotide identity. Antonie van Leeuwenhoek 110, 1281 - 1286.).

[0103] To further clarify the phylogenetic status of the strain, representative strains of all species in the genus Saccharibacillus and 16S rRNA gene sequences of related species in the family Paenibacillaceae were selected to construct a phylogenetic tree( Figure 3 ).

[0104] In summary, the strain S22909 of the present invention has some obvious characteristic differences from the existing species in the genus Saccharibacillus, including aspects such as phenotype, physiological and biochemical properties, and cytochemical components. At the same time, phylogenetic analysis at the gene level further shows that strain S22909 can be distinguished from the existing valid species in the genus Saccharibacillus, fully proving that the strain S22909 of the present invention represents a new species in the genus Saccharibacillus, named Saccharibacillus phosphatilytica. At the same time, through the detection of alkaline phosphatase activity, it is also proved that the strain of the present invention has alkaline phosphatase activity, which can convert insoluble phosphorus in the soil into soluble phosphorus for plant absorption and utilization, and has broad application prospects in degrading organic phosphorus and promoting plant growth; at the same time, the strain has the ability to degrade cellulose and has broad application prospects in cellulose degradation. Therefore, the strain of the present invention can be used in the future for the development of various aspects such as phosphorus - solubilizing bacterial fertilizers, environmental governance, soil improvement production, and biofuel development.

[0105] Saccharibacillus phosphatilytica S22909 was deposited at the China General Microbiological Culture Collection Center on July 7, 2022, with the deposit number CGMCC NO. 25247.

[0106] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any changes, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. Bacillus amyloliquefaciens Saccharibacillus phosphatilytica ) S22909, with the registration number of CGMCC No. 25247 at the China General Microbiological Culture Collection Center.

2. The culture of Bacillus amylolyticus phosphaticum ( Saccharibacillus phosphatilytica ) S22909 as claimed in claim 1 is a substance obtained by culturing Bacillus amylolyticus phosphaticum ( Saccharibacillus phosphatilytica ) S22909 in a bacterial culture medium.

3. Bacterial agent, characterized in that: The bacterial agent contains the phosphate-solubilizing Bacillus glycines ( Saccharibacillus phosphatilytica ) S22909 or the culture as described in claim 2.

4. The microbial agent according to claim 3, characterized in that: The microbial agent is a microbial agent for degrading cellulose and / or degrading organic phosphorus.

5. Use of Bacillus amylolyticus phosphaticum ( Saccharibacillus phosphatilytica ) S22909, the culture according to claim 2, or the bacterial agent according to claim 3 or 4 in any of the following: (A1) Degrading cellulose; (A2) Preparing a product for degrading cellulose; (A3) Preparing cellulase; (A4) Preparing a product with cellulase activity; (A5) Preparing a product with endoglucanase activity; (A6) Degrading organic phosphorus; (A7) Preparing a product for degrading organic phosphorus; (A8) Preparing a product with alkaline phosphatase activity.

6. Use of Bacillus amylolyticus phosphaticum ( Saccharibacillus phosphatilytica ) S22909 or the culture according to claim 2 or the microbial inoculum according to claim 3 or 4 in any of the following: (B1) Promoting plant growth; (B2) Developing a phosphorus-solubilizing bacterial fertilizer; (B3) Soil improvement.

7. A product for degrading cellulose, the active ingredient of which is the phosphate-solubilizing Bacillus licheniformis ( Saccharibacillus phosphatilytica ) S22909 described in claim 1, or the culture described in claim 2, or the bacterial agent described in claim 3 or 4.

8. A product with cellulase activity, and its active ingredient is the phosphate-solubilizing Bacillus licheniformis ( Saccharibacillus phosphatilytica ) S22909 described in claim 1, or the culture described in claim 2, or the microbial inoculum described in claim 3 or 4.

9. A product with endoglucanase activity, and its active ingredient is the phosphate-solubilizing Bacillus licheniformis ( Saccharibacillus phosphatilytica ) S22909 described in claim 1, or the culture described in claim 2, or the microbial inoculum described in claim 3 or 4.

10. A product for degrading organophosphorus, the active ingredient of which is the phosphorus-solubilizing Bacillus glycines ( Saccharibacillus phosphatilytica ) S22909 or the culture described in claim 2 or the microbial agent described in claim 3 or 4.

11. A product with alkaline phosphatase activity, the active ingredient of which is the phosphate-solubilizing Bacillus glycinifermentans ( Saccharibacillus phosphatilytica ) S22909 described in claim 1, or the culture described in claim 2, or the bacterial agent described in claim 3 or 4.

12. A method for degrading cellulose, comprising the following steps: treating a sample to be treated with the Bacillus phosphaticum Saccharibacillus phosphatilytica S22909 described in claim 1, or the culture described in claim 2, or the bacterial agent described in claim 3 or 4.

13. A method for degrading organophosphorus, comprising the following steps: treating a sample to be treated with the Bacillus saccharolyticus Saccharibacillus phosphatilytica S22909 described in claim 1, or the culture described in claim 2, or the microbial agent described in claim 3 or 4.

14. Use of Bacillus amylolyticus S22909 as claimed in claim 1 in the preparation of metabolites or the culture as claimed in claim 2 or the microbial agent as claimed in claim 3 or 4. Saccharibacillus phosphatilytica ​

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